Method of using reduced dimensionality nuclear magnetic resonance spectroscopy for rapid chemical shift assignment and secondary structure determination of proteins

ABSTRACT

The present invention discloses eight new reduced dimensionality (RD) triple resonance nuclear magnetic resonance (NMR) experiments for measuring chemical shift values of certain nuclei in a protein molecule. The RD 3D  HA , CA ,(CO),N,HN NMR experiment and the RD 3D  H , C ,(C-TOCSY-CO),N,HN NMR experiment are designed to yield “sequential” connectivities, while the RD 3D  H   αβ , C   αβ ,CO,HA NMR experiment and the RD 3D  H   αβ , C   αβ ,HN NMR experiment provide “intraresidue” connectivities. The RD 3D  H , C ,C,H-COSY NMR experiment, the RD 3D  H , C ,C,H-TOCSY NMR experiment, and the RD 2D  H , C ,H-COSY NMR experiment allow one to obtain assignments for aliphatic and aromatic side chain chemical shifts, while the RD 2D  HB,CB ,(CG,CD),HD NMR experiment provide information for the aromatic side chain chemical shifts. In addition, a method of conducting suites of RD triple resonance NMR experiments for high-throughput resonance assignment of proteins and identification of the location of secondary structure elements are disclosed.

[0001] The present invention claims the benefit of U.S. ProvisionalPatent Application Serial No. 60/215,649, filed Jun. 30, 2000, which ishereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to methods of using reduceddimensionality nuclear magnetic resonance (NMR) spectroscopy forobtaining chemical shift assignment and structure determination ofproteins.

BACKGROUND OF THE INVENTION

[0003] The use of triple resonance (TR) nuclear magnetic resonance (NMR)experiments for the resonance assignment of polypeptide chains viaheteronuclear scalar connectivities (Montelione et al., J. Am Chem.Soc., 111:5474-5475 (1989); Montelione et al., J. Magn. Reson.,87:183-188 (1989); Kay et al., J. Magn. Reson., 89:496-514 (1990); Ikuraet al., Biochemistry, 29:4659-8979 (1990); Edison et al., MethodsEnzymol., 239:3-79 (1994)) is a standard approach which neatlycomplements the assignment protocol based on ¹H—¹H nuclear Overhausereffects (NOE) (Wüthrich, NMR of Proteins and Nucleic Acids, Wiley, NewYork (1986)). In addition, triple resonance NMR spectra are highlyamenable to a fast automated analysis (Friedrichs et al., J. Biomol.NMR, 4:703-726 (1994); Zimmerman et al., J. Biomol. NMR, 4:241-256(1994); Bartels et al., J. Biomol. NMR, 7:207-213 (1996); Morelle etal., J. Biomol. NMR, 5:154-160 (1995); Buchler et al., J. Magn. Reson.,125:34-42 (1997); Lukin et al., J. Biomol. NMR, 9:151-166 (1997)),yielding the ¹³C^(α/β) chemical shifts at an early stage of theassignment procedure. This enables both, the identification of regularsecondary structure elements without reference to NOEs (Spera et al., J.Am. Chem. Soc., 113: 5490-5491 (1991)) and the derivation of (φ,ψ)-angleconstraints which serve to reduce the number of cycles consisting ofnuclear Overhauser enhancement spectroscopy (NOESY) peak assignment andstructure calculation (Luginbühl et al., J. Magn. Reson., B 109:229-233(1995)).

[0004] NMR assignments are prerequisite for NMR-based structural biology(Wüthrich, NMR of Proteins and Nucleic Acid, Wiley:New York (1986)) and,thus, for high-throughput (HTP) structure determination in structuralgenomics (Rost, Structure, 6:259-263 (1998); Montelione et al., NatureStruct. Biol., 6:11-12. (1999); Burley, Nature Struc Biol., 7:932-934(2000)) and for exploring structure-activity relationships (SAR) by NMRfor drug discovery (Shuker et al., Science, 274:1531-1534 (1996)). Theaims of structural genomics are to (i) explore the naturally occurring“protein fold space” and (ii) contribute to the characterization offunction through the assignment of atomic resolution three-dimensional(3D) structures to proteins. It is now generally acknowledged that NMRwill play an important role in structural genomics (Montelione et al.,Nature Struc. Biol., 7:982-984 (2000)). The resulting demand for HTPstructure determination requires fast and automated NMR data collectionand analysis protocols (Moseley et al., Curr. Opin. Struct. Biol.,9:635-642 (1999)).

[0005] The establishment of a HTP NMR structural genomics pipelinerequires two key objectives in data collection. Firstly, the measurementtime should be minimized in order to (i) lower the cost per structureand (ii) relax the constraint that NMR samples need to be stable over along period of measurement time. The recent introduction of commercialcryogenic probes (Styles et al., J. Magn. Reson., 60:397-404 (1984);Flynn et al., J. Am Chem. Soc., 122:4823-4824 (2000)) promises to reducemeasurement times by about a factor of ten or more, and will greatlyimpact the realization of this first objective. Secondly, reliableautomated spectral analysis requires recording of a “redundant” set ofmultidimensional NMR experiments each affording good resolution (whichrequires appropriately long maximal evolution times in all indirectdimensions). Concomitantly, it is desirable to keep the total number ofNMR spectra small in order to minimize “interspectral” variations ofchemical shift measurements, which may impede automated spectralanalysis. Straightforward consideration of this second objective wouldsuggest increasing the dimensionality of the spectra, preferably byimplementing a suite of four- or even higher-dimensional NMRexperiments. Importantly, however, the joint realization of the firstand second objectives is tightly limited by the rather large lowerbounds of higher-dimensional TR NMR measurement times if appropriatelylong maximal evolution times are chosen.

[0006] Hence, “sampling limited” and “sensitivity limited” datacollection regimes are distinguished, depending on whether the samplingof the indirect dimensions or the sensitivity of the multidimensionalNMR experiments “per se” determines the minimally achievable measurementtime. As a matter of fact, the ever increasing performance of NMRspectrometers will soon lead to the situation where, for many proteinsamples, the sensitivity of the NMR spectrometers do not constitute theprime bottleneck determining minimal measurement times. Instead, theminimal measurement times encountered for recording conventionalhigher-dimensional NMR schemes will be “sampling limited,” particularlyas high sensitivity cryoprobes become generally available. As structuredeterminations of proteins rely on nearly complete assignment ofchemical shifts, which are obtained using multidimensional ¹³C, ¹⁵N,¹H-TR NMR experiments (Montelione et al., J. Am Chem. Soc.,111:5474-5475 (1989); Montelione, et al., J. Magn. Reson., 87:183-188(1989); Ikura et al., Biochemistry, 29:4659-8979 (1990)), thedevelopment of TR NMR techniques that avoid the sampling limited regimerepresents a key challenge for future biomolecular NMR methodsdevelopment.

[0007] Reduced dimensionality (RD) TR NMR experiments (Szyperski et al.,J. Biomol. NMR, 3:127-132 (1993); Szyperski et al., J. Am. Chem. Soc.,115:9307-9308 (1993); Szyperski et al., J. Magn. Reson., B 105:188-191(1994); Brutscher et al., J. Magn. Reson., B 105:77-82 (1994); Szyperskiet al., J. Magn. Reson., B 108: 197-203 (1995); Brutscher et al., J.Biomol. NMR, 5:202-206 (1995); Löhr et al., J. Biomol. NMR, 6:189-197(1995); Szyperski et al., J. Am. Chem. Soc., 118:8146-8147 (1996);Szyperski et al., J. Magn. Reson., 28:228-232 (1997); Bracken et al., J.Biomol. NMR, 9:94-100 (1997); Sklenar et al., J. Magn. Reson.,130:119-124 (1998); Szyperski et al., J. Biomol. NMR, 11:387-405(1998)), designed for simultaneous frequency labeling of two spin typesin a single indirect dimension, offer a viable strategy to circumventrecording NMR spectra in a sampling limited fashion. RD NMR is based ona projection technique for reducing the spectral dimensionality of TRexperiments: the chemical shifts of the projected dimension give rise toa cosine-modulation of the transfer amplitude, yielding peak doubletsencoding n chemical shifts in a n-1 dimensional spectrum (Szyperski etal., J. Biomol. NMR, 3:127-132 (1993); Szyperski et al., J. Am. Chem.Soc., 115:9307-9308 (1993)). As a key result, this allows recordingprojected four-dimensional (4D) NMR experiments with maximal evolutiontimes typically achieved in the corresponding conventional 3D NMRexperiments (Szyperski et al., J. Biomol. NMR, 3:127-132 (1993);Szyperski et al., J. Am. Chem. Soc., 115:9307-9308 (1993); Szyperski etal.,. J. Magn. Reson. B 105:188-191 (1994); Szyperski et al., J. Magn.Reson., B 108: 197-203 (1995); Szyperski et al., J. Am. Chem. Soc.,118:8146-8147 (1996); Szyperski et al., J. Magn. Reson., 28:228-232(1997); Bracken et al., J. Biomol. NMR, 9:94-100 (1997); Sklenar et al.,J. Magn. Reson., 130:119-124 (1998); Szyperski et al., J. Biomol. NMR,11:387-405 (1998)). Furthermore, axial coherences, arising from eitherincomplete insensitive nuclei enhanced by polarization transfer (INEPT)or heteronuclear magnetization, can be observed as peaks located at thecenter of the doublets (Szyperski et al., J. Am. Chem. Soc.,118:8146-8147 (1996)). This allows both the unambiguous assignment ofmultiple doublets with degenerate chemical shifts in the otherdimensions and the identification of cross peak pairs by symmetrizationof spectral strips about the position of the central peak (Szyperski etal., J. Am. Chem. Soc., 118:8146-8147 (1996); Szyperski et al., J.Biomol. NMR, 11:387-405 (1998)). Hence, observation of central peaks notonly restores the dispersion of the parent, higher-dimensionalexperiment, but also provides access to reservoir of axial peakmagnetization (Szyperski et al., J. Am. Chem. Soc., 118:8146-8147(1996)). Historically, RD NMR experiments were first designed tosimultaneously recruit both ¹H and heteronuclear magnetization(Szyperski et al., J. Am. Chem. Soc., 118:8146-8147 (1996)) for signaldetection, a feature that has also gained interest for improvingtransverse relaxation-optimized spectroscopy (TROSY) pulse schemes(Pervushin et al., Proc. Natl. Acad. Sci. USA, 94:12366-12371 (1997);Salzmann et al., J. Am. Chem. Soc., 121:844-848 (1999); Pervushin etal., J. Biomol. NMR, 12:345-348, (1998)). Moreover, RD two-spincoherence NMR spectroscopy (Szyperski et al., J. Biomol. NMR, 3:127-132(1993)) subsequently also called zero-quantum/double-quantum (ZQ/DQ) NMRspectroscopy (Rexroth et al., J. Am. Chem. Soc., 17:10389-10390 (1995)),served as a valuable radio-frequency (r.f.) pulse module for measurementof scalar coupling constants (Rexroth et al., J. Am. Chem. Soc., 17:10389-10390 (1995)) and cross-correlated heteronuclear relaxation (Reifet al., Science, 276:1230-1233 (1997); Yang et al., J. Am. Chem. Soc.,121:3555-3556 (1999); Chiarparin et al., J. Am. Chem. Soc.,122:1758-1761 (2000); Brutscher et al., J. Magn. Reson., 130:346-351(1998); Brutscher, Concepts Magn. Reson., 122:207-229 (2000)).

[0008] The present invention is directed to overcoming the deficienciesin the art.

SUMMARY OF THE INVENTION

[0009] The present invention relates to a method of conducting a reduceddimensionality three-dimensional (3D) HA,CA,(CO),N,HN nuclear magneticresonance (NMR) experiment by measuring the chemical shift values forthe following nuclei of a protein molecule having two consecutive aminoacid residues, i−1 and i: (1) an α-proton of amino acid residue i−1,¹H^(α) _(i−1); (2) an α-carbon of amino acid residue i−1, ¹³C^(α)_(i−1); (3) a polypeptide backbone amide nitrogen of amino acid residuei, ¹⁵N_(i); and (4) a polypeptide backbone amide proton of amino acidresidue i, ¹H^(N) _(i). The method involves providing a protein sampleand applying radiofrequency pulses to the protein sample which effect anuclear spin polarization transfer where the chemical shift evolutionsof ¹H^(α) _(i−1) and ¹³C^(α) _(l−1) of amino acid residue i−1 areconnected to the chemical shift evolutions of ¹⁵N, and ¹H^(N) _(i) ofamino acid residue i, under conditions effective (1) to generate NMRsignals encoding the chemical shift values of ¹³C^(α) _(i−1) and ¹⁵N, ina phase sensitive manner in two indirect time domain dimensions,t₁(¹³C^(α)) and t₂(¹⁵N), respectively, and the chemical shift value of¹H^(N) _(i) in a direct time domain dimension, t₃(¹H^(N)), and (2) tocosine modulate the ¹³C^(a) _(i−1) chemical shift evolution int₁(¹³C^(α)) with the chemical shift evolution of ¹H^(α) _(i−1). Then,the NMR signals are processed to generate a 3D NMR spectrum with aprimary peak pair derived from the cosine modulating, where (1) thechemical shift values of ¹⁵N_(l) and ¹H^(N) _(l) are measured in twofrequency domain dimensions, ω₂(¹⁵N) and ω₃(¹H^(N)), respectively, and(2) the chemical shift values of ¹H^(α) _(i−1) and ¹³C^(α) _(i−1) aremeasured in a frequency domain dimension, ω₁(¹³C^(α)), by the frequencydifference between the two peaks forming the primary peak pair and thefrequency at the center of the two peaks, respectively.

[0010] The present invention also relates to a method of conducting areduced dimensionality three-dimensional (3D) H,C,(C-TOCSY-CO),N,HNnuclear magnetic resonance (NMR) experiment by measuring the chemicalshift values for the following nuclei of a protein molecule having twoconsecutive amino acid residues, i−1 and i: (1) aliphatic protons ofamino acid residue i−1, ¹H^(ali) _(l−1); (2) aliphatic carbons of aminoacid residue i−1, ¹³C^(ali) _(i−1); (3) a polypeptide backbone amidenitrogen of amino acid residue i, ¹⁵N_(l); and (4) a polypeptidebackbone amide proton of amino acid residue i, ¹H^(N) _(l). The methodinvolves providing a protein sample and applying radiofrequency pulsesto the protein sample which effect a nuclear spin polarization transferwhere the chemical shift evolutions of ¹H^(ali) _(i−1) and ¹³C^(ali)_(l−1) of amino acid residue i−1 are connected to the chemical shiftevolutions of ¹⁵N_(i) and ¹H^(N) _(i) of amino acid residue i, underconditions effective (1) to generate a NMR signal encoding the chemicalshifts of ¹³C^(ali) _(i−1) and ¹⁵N_(i) in a phase sensitive manner intwo indirect time domain dimensions, t₁(¹³C^(ali)) and t₂(¹⁵N),respectively, and the chemical shift of ¹H^(N) _(i) in a direct timedomain dimension, t₃(¹H^(N)), and (2) to cosine modulate the chemicalshift evolutions of ¹³C^(ali) _(i−1) in t₁(¹³C^(ali)) with the chemicalshift evolutions of ¹H^(ali) _(i−1). Then, the NMR signals are processedto generate a 3D NMR spectrum with peak pairs derived from the cosinemodulating where (1) the chemical shift values of ¹⁵N_(l) and ¹H^(N)_(i) are measured in two frequency domain dimensions, ω₂(¹⁵N) andω₃(¹H^(N)), respectively, and (2) the chemical shift values of ¹H^(ali)_(i−1) and ¹³C^(ali) _(i−1) are measured in a frequency domaindimension, ω₁(₁₃C^(ali)), by the frequency differences between the twopeaks forming the peak pairs and the frequencies at the center of thetwo peaks, respectively.

[0011] Another aspect of the present invention relates to a method ofconducting a reduced dimensionality three-dimensional (3D) H ^(α/β),C^(α/β),CO,HA nuclear magnetic resonance (NMR) experiment by measuringthe chemical shift values for the following nuclei of a protein moleculehaving an amino acid residue, i: (1) a β-proton of amino acid residue i,¹H^(β) _(i); (2) a β-carbon of amino acid residue i, ¹³C^(β) _(i); (3)an α-proton of amino acid residue i, ¹H^(α) _(i); (4) an α-carbon ofamino acid residue i, ¹³C^(β) _(i); and (5) a polypeptide backbonecarbonyl carbon of amino acid residue i, ¹³C′_(i). The method involvesproviding a protein sample and applying radiofrequency pulses to theprotein sample which effect a nuclear spin polarization transfer wherethe chemical shift evolutions of ¹H^(α) _(l), ¹H^(β) _(l), ¹³C^(α) _(l),and ¹³C^(β) _(l) are connected to the chemical shift evolution of¹³C′_(l), under conditions effective (1) to generate NMR signalsencoding the chemical shift values of ¹³C^(α) _(l), ¹³C^(β) _(l) and¹³C′_(l) in a phase sensitive manner in two indirect time domaindimensions, t₁(¹³C^(α/β)) and t₂(¹³C′), respectively, and the chemicalshift value of ¹H^(α) _(i) in a direct time domain dimension, t₃(¹H^(α)), and (2) to cosine modulate the chemical shift evolutions of¹³C^(α) _(i) and ¹³C^(β) _(i) in t₁(¹³C^(α/β)) with the chemical shiftevolutions of ¹H^(α) _(l), and ¹H^(β) _(i), respectively. Then, the NMRsignals are processed to generate a 3D NMR spectrum with peak pairsderived from the cosine modulating where (1) the chemical shift valuesof ¹³C′_(i) and ¹H^(α) _(i) are measured in two frequency domaindimensions, ω₂(¹³C′) and ω₃(¹H^(α)), respectively, and (2) (i) thechemical shift values of ¹H^(α) _(i) and ¹H^(β) _(i) are measured in afrequency domain dimension, ω₁(¹³C^(α/β)), by the frequency differencesbetween the two peaks forming the peak pairs, and (ii) the chemicalshift values of ¹³C^(α) _(i), and ¹³C^(β) _(l) are measured in afrequency domain dimension, ω₁(¹³C^(α/β)), by the frequencies at thecenter of the two peaks forming the peak pairs.

[0012] A further aspect of the present invention relates to a method ofconducting a reduced dimensionality three-dimensional (3D) H ^(α/β),C^(α/β),N,HN nuclear magnetic resonance (NMR) experiment by measuring thechemical shift values for the following nuclei of a protein moleculehaving an amino acid residue, i: (1) a β-proton of amino acid residue i,¹H^(β) _(i); (2) a β-carbon of amino acid residue i, ¹³C^(β) _(i); (3)an α-proton of amino acid residue i, ¹H^(α) _(i); (4) an α-carbon ofamino acid residue i, ¹³C^(α) _(i); (5) a polypeptide backbone amidenitrogen of amino acid residue i, ¹⁵N_(i); and (6) a polypeptidebackbone amide proton of amino acid residue i, ¹H^(N) _(l). The methodinvolves providing a protein sample and applying radiofrequency pulsesto the protein sample which effect a nuclear spin polarization transferwhere the chemical shift evolutions of ¹H^(α) _(i), ¹H^(β) _(i), ¹³C^(α)_(i), and ¹³C^(β) _(l) are connected to the chemical shift evolutions of¹⁵N_(i) and ¹H^(N) _(i), under conditions effective (1) to generate NMRsignals encoding the chemical shift values of ¹³C^(α) _(i), ¹³C^(β) _(i)and ¹⁵N_(l) in a phase sensitive manner in two indirect time domaindimensions, t₁(¹³C^(α/β)) and t₂(¹⁵N), respectively, and the chemicalshift value of ¹H^(N) _(i) in a direct time domain dimension,t₃(¹H^(N)), and (2) to cosine modulate the chemical shift evolutions of¹³C^(α) _(i) and ¹³C^(β) _(i) in t₁(¹³C^(α/β)) with the chemical shiftevolutions of ¹H^(α) _(l) and ¹H^(β) _(l), respectively. Then, the NMRsignals are processed to generate a 3D NMR spectrum with peak pairsderived from the cosine modulating where (1) the chemical shift valuesof ¹⁵N_(i) and ¹H^(N) _(i) are measured in two frequency domaindimensions, ω₂(¹⁵N) and ω₃(¹H^(N)), respectively, and (2) (i) thechemical shift values of ¹H^(α) _(i) and ¹H^(β) _(i) are measured in afrequency domain dimension, ω₁(¹³C^(α/β)), by the frequency differencesbetween the two peaks forming the peak pairs, and (ii) the chemicalshift values of ¹³C^(α) _(l), and ¹³C^(β) _(i) are measured in afrequency domain dimension, ω₁(¹³C^(α/β)), by the frequencies at thecenter of the two peaks forming the peak pairs.

[0013] The present invention also relates to a method of conducting areduced dimensionality three-dimensional (3D) H,C,C,H-COSY nuclearmagnetic resonance (NMR) experiment by measuring the chemical shiftvalues for ¹H^(m), ¹³C^(m), ¹H^(n), and ¹³C^(n) of a protein moleculewhere m and n indicate atom numbers of two CH, CH₂ or CH₃ groups thatare linked by a single covalent carbon—carbon bond in an amino acidresidue. The method involves providing a protein sample and applyingradiofrequency pulses to the protein sample which effects a nuclear spinpolarization transfer where the chemical shift evolutions of ¹H^(m) and¹³C^(m) are connected to the chemical shift evolutions of ¹H^(n) and¹³C^(n), under conditions effective (1) to generate NMR signals encodingthe chemical shift values of ¹³C^(m) and ¹³C^(n) in a phase sensitivemanner in two indirect time domain dimensions, t₁(¹³C^(m)) andt₂(¹³C^(n)), respectively, and the chemical shift value of ¹H^(n) in adirect time domain dimension, t₃(¹H^(n)), and (2) to cosine modulate thechemical shift evolution of ¹³C^(m) in t₁(¹³C^(m)) with the chemicalshift evolution of ¹H_(m). Then, the NMR signals are processed togenerate a 3D NMR spectrum with peak pairs derived from the cosinemodulating where (1) the chemical shift values of ¹³C^(n) and ¹H^(n) aremeasured in two frequency domain dimensions, ω₂(¹³C^(n)) and ω₃(¹H^(n)),respectively, and (2) the chemical shift values of ¹H^(m) and ¹³C^(m)are measured in a frequency domain dimension, ω₁(¹³C^(m)), by thefrequency differences between the two peaks forming the peak pairs andthe frequencies at the center of the two peaks, respectively.

[0014] Another aspect of the present invention relates to a method ofconducting a reduced dimensionality three-dimensional (3D) H,C,C,H-TOCSYnuclear magnetic resonance (NMR) experiment by measuring the chemicalshift values for ¹H^(m), ¹³C^(m), ¹H^(n), and ¹³C^(n) of a proteinmolecule where m and n indicate atom numbers of two CH, CH₂ or CH₃groups that may or may not be directly linked by a single covalentcarbon-carbon bond in an amino acid residue. The method involvesproviding a protein sample and applying radiofrequency pulses to theprotein sample which effect a nuclear spin polarization transfer wherethe chemical shift evolutions of ¹H^(m) and ¹³C^(m) are connected to thechemical shift evolutions of ¹H^(n) and ¹³C^(n), under conditionseffective (1) to generate NMR signals encoding the chemical shift valuesof ¹³C^(m) and ¹³C^(n) in a phase sensitive manner in two indirect timedomain dimensions, t₁(¹³C^(m)) and t₂(¹³C^(n)), and the chemical shiftvalue of ¹H^(n) in a direct time domain dimension, t₃(¹H^(n)), and (2)to cosine modulate the chemical shift evolution of ¹³C^(m) int₁(¹³C^(m)) with the chemical shift evolution of ¹H^(m). Then, the NMRsignals are processed to generate a 3D NMR spectrum with peak pairsderived from the cosine modulating where (1) the chemical shift valuesof ¹³C^(n) and ¹H^(n) are measured in two frequency domain dimensions,ω₂(¹³C^(n)) and ω₃(¹H^(n)), respectively, and (2) the chemical shiftvalues of ¹H^(m) and ¹³C^(m) are measured in a frequency domaindimension, ω₁(¹³C^(m)), by the frequency differences between the twopeaks forming the peak pairs and the frequencies at the center of thetwo peaks, respectively.

[0015] A further aspect of the present invention relates to a method ofconducting a reduced dimensionality two-dimensional (2D)HB,CB,(CG,CD),HD nuclear magnetic resonance (NMR) experiment bymeasuring the chemical shift values for the following nuclei of aprotein molecule: (1) a β-proton of an amino acid residue with anaromatic side chain, ¹H^(β); (2) a β-carbon of an amino acid residuewith an aromatic side chain, ¹³C^(β); and (3) a δ-proton of an aminoacid residue with an aromatic side chain, ¹H^(δ). The method involvesproviding a protein sample and applying radiofrequency pulses to theprotein sample which effect a nuclear spin polarization transfer wherethe chemical shift evolutions of ¹H^(β) and ¹³C^(β) are connected to thechemical shift evolution of ¹H^(δ), under conditions effective (1) togenerate NMR signals encoding the chemical shift value of ¹³C^(β) in aphase sensitive manner in an indirect time domain dimension,t₁(¹³C^(β)), and the chemical shift value of ¹H^(δ) in a direct timedomain dimension, t₂(¹H^(δ)), and (2) to cosine modulate the chemicalshift evolution of ¹³C^(β)in t₁(¹³C^(β)) with the chemical shiftevolution of ¹H^(β). Then, the NMR signals are processed to generate a2D NMR spectrum with a peak pair derived from the cosine modulatingwhere (1) the chemical shift value of ¹H^(δ) is measured in a frequencydomain dimension, ω₂(¹H^(δ)), and (2) the chemical shift values of¹H^(β) and ¹³C^(β) are measured in a frequency domain dimension,ω₁(¹³C^(β)), by the frequency difference between the two peaks formingthe peak pair and the frequency at the center of the two peaks,respectively.

[0016] The present invention also relates to a method of conducting areduced dimensionality two-dimensional (2D) H,C,H-COSY nuclear magneticresonance (NMR) experiment by measuring the chemical shift values for¹H^(m), ¹³C^(m), and ¹H^(n) of a protein molecule where m and n indicateatom numbers of two CH, CH₂ or CH₃ groups in an amino acid residue. Themethod involves providing a protein sample and applying radiofrequencypulses to the protein sample which effect a nuclear spin polarizationtransfer where the chemical shift evolutions of ¹H^(m) and ¹³C^(m) areconnected to the chemical shift evolution of ¹H^(n), under conditionseffective (1) to generate NMR signals encoding the chemical shift valueof ¹³C^(m) in a phase sensitive manner in an indirect time domaindimension, t₁(¹³C^(m)), and the chemical shift value of ¹H^(n) in adirect time domain dimension, t₂(¹H^(n)), and (2) to cosine modulate thechemical shift evolution of ¹³C^(m) in t₁(¹³C^(m)) with the chemicalshift evolution of ¹H^(m). Then, the NMR signals are processed togenerate a 2D NMR spectrum with peak pairs derived from the cosinemodulating where (1) the chemical shift value of ¹H^(n) is measured in afrequency domain dimension, ω₂(¹H^(n)), and (2) the chemical shiftvalues of ¹H^(m) and ¹³C^(m) are measured in a frequency domaindimension, ω₁(¹³C^(m)), by the frequency differences between the twopeaks forming the peak pairs and the frequencies at the center of thetwo peaks, respectively.

[0017] Another aspect of the present invention relates to a method forsequentially assigning chemical shift values of an α-proton, ¹H^(α), anα-carbon, ¹³C^(α), a polypeptide backbone amide nitrogen, ¹⁵N, and apolypeptide backbone amide proton, ¹H^(N), of a protein molecule. Themethod involves providing a protein sample and conducting a set ofreduced dimensionality (RD) nuclear magnetic resonance (NMR) experimentson the protein sample including: (1) a RD three-dimensional (3D)HA,CA,(CO),N,HN NMR experiment to measure and connect chemical shiftvalues of the α-proton of amino acid residue i−1, ¹H^(α) _(l−1), theα-carbon of amino acid residue i−1, ¹³C^(α) _(i−1), the polypeptidebackbone amide nitrogen of amino acid residue i, ¹⁵N_(i), and thepolypeptide backbone amide proton of amino acid residue i, ^(l)H^(N)_(i) and (2) a RD 3D HNNCAHA NMR experiment to measure and connect thechemical shift values of the α-proton of amino acid residue i, ¹H^(α)_(i), the α-carbon of amino acid residue i, ¹³C^(α) _(i), ¹⁵N_(i), and¹H^(N) _(l). Then, sequential assignments of the chemical shift valuesof ¹H^(α), ¹³C^(α), ¹⁵N, and ¹H^(N) are obtained by (i) matching thechemical shift values of ¹H^(α) _(i−1) and ¹³C^(α) _(i−1) with thechemical shift values of ¹H^(α) _(i) and ¹³C^(α) _(i), (ii) using thechemical shift values of ¹H^(α) _(i−1) and ¹³C^(α) _(i−1) to identifythe type of amino acid residue i−1, and (iii) mapping sets ofsequentially connected chemical shift values to the amino acid sequenceof the polypeptide chain and using the chemical shift values to locatesecondary structure elements within the polypeptide chain.

[0018] Yet another aspect of the present invention relates to a methodfor sequentially assigning chemical shift values of a β-proton, ¹H^(β),a β-carbon, ¹³C^(β), an α-proton, ¹H^(α), an α-carbon, ¹³C^(α), apolypeptide backbone amide nitrogen, ¹⁵N, and a polypeptide backboneamide proton, ¹H^(N), of a protein molecule. The method involvesproviding a protein sample and conducting a set of reduceddimensionality (RD) nuclear magnetic resonance (NMR) experiments on theprotein sample including: (1) a RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMRexperiment to measure and connect the chemical shift values of theβ-proton of amino acid residue i−1, ¹H^(β) _(l−1), the β-carbon of aminoacid residue i−1, ¹³C^(β) _(l−1), the α-proton of amino acid residuei−1, ¹H^(α) _(l−1), the α-carbon of amino acid residue i−1, ¹³C^(β)_(l−1), the polypeptide backbone amide nitrogen of amino acid residue i,¹⁵N_(i), and the polypeptide backbone amide proton of amino acid residuei, ¹H^(N) _(i) and (2) a RD 3D H ^(α/β), C ^(α/β),N,HN NMR experiment tomeasure and connect the chemical shift values of the β-proton of aminoacid residue i, ¹H^(β) _(i), the β-carbon of amino acid residue i,¹³C^(β) _(i), the α-proton of amino acid residue i, ¹H^(α) _(i), theα-carbon of amino acid residue i, ¹³C^(α) _(i), ¹⁵N_(l), and ¹H^(N)_(l). Then, sequential assignments of the chemical shift values of¹H^(β), ¹³C^(β), ¹H^(α), ¹³C^(α), ¹⁵N, and ¹H^(N) are obtained by (i)matching the chemical shift values of the α- and β-protons of amino acidresidue i−1, ¹H^(α/β) _(i−1), and the α- and β-carbons of amino acidresidue i−1, ¹³C^(α/β) _(i−1), with the chemical shift values of¹H^(α/β) _(i) and ¹³C^(α/β) _(i), (ii) using the chemical shift valuesof ¹H^(α/β) _(i−1) and ¹³C^(α/β) _(i−1) to identify the type of aminoacid residue i−1, and (iii) mapping sets of sequentially connectedchemical shift values to the amino acid sequence of the polypeptidechain and using the chemical shift values to locate secondary structureelements within the polypeptide chain.

[0019] A further aspect of the present invention involves a method forsequentially assigning chemical shift values of aliphatic protons,¹H^(ali), aliphatic carbons, ¹³C^(ali), a polypeptide backbone amidenitrogen, ¹⁵N, and a polypeptide backbone amide proton, ¹H^(N), of aprotein molecule. The method involves providing a protein sample andconducting a set of reduced dimensionality (RD) nuclear magneticresonance (NMR) experiments on the protein sample including: (1) a RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment to measure and connect the chemicalshift values of the aliphatic protons of amino acid residue i−1,¹H^(ali) _(i−1) , the aliphatic carbons of amino acid residue i−1,¹³C^(ali) _(i−1), the polypeptide backbone amide nitrogen of amino acidresidue i, ¹⁵N_(i), and the polypeptide backbone amide proton of aminoacid residue i, ¹H^(N) _(i) and (2) a RD 3D H ^(α/β),C ^(α/β),N,HN NMRexperiment to measure and connect the chemical shift values of theβ-proton of amino acid residue i, ¹H^(β) _(i), the β-carbon of aminoacid residue i, ¹³C^(β) _(l), the α-proton of amino acid residue i,¹H^(α) _(l), the α-carbon of amino acid residue i, ¹³C^(α) _(i),¹⁵N_(l), and ¹H^(N) _(l). Then, sequential assignments of the chemicalshift values of ¹H^(ali), ¹³C^(ali), ¹⁵N, and ¹H^(N) are obtained by (i)matching the chemical shift values of the α- and β-protons of amino acidresidue i−1, ¹H^(α/β) _(i−1), and the α- and β-carbons of amino acidresidue i−1, ¹³C^(α/β) _(i−1), with the chemical shift values of¹H^(α/β) _(l) and ¹³C^(α/β) _(i) of amino acid residue i, (ii) using thechemical shift values of ¹H^(ali) _(l−1) and ¹³C^(ali) _(l−1) toidentify the type of amino acid residue i−1, and (iii) mapping sets ofsequentially connected chemical shift values to the amino acid sequenceof the polypeptide chain and using the chemical shift values to locatesecondary structure elements within the polypeptide chain.

[0020] The present invention also relates to a method for sequentiallyassigning chemical shift values of aliphatic protons, ¹H^(ali),aliphatic carbons, ¹³C^(ali), a polypeptide backbone amide nitrogen,¹⁵N, and a polypeptide backbone amide proton, ¹H^(N), of a proteinmolecule. The method involves providing a protein sample and conductinga set of reduced dimensionality (RD) nuclear magnetic resonance (NMR)experiments on the protein sample including: (1) a RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment to measure and connect the chemicalshift values of the aliphatic protons of amino acid residue i−1,¹H^(ali) _(i−1), the aliphatic carbons of amino acid residue i−1,¹³C^(ali) _(i−1), the polypeptide backbone amide nitrogen of amino acidresidue i, ¹⁵N_(i), and the polypeptide backbone amide proton of aminoacid residue i, ¹H^(N) _(i) and (2) a RD 3D HNNCAHA NMR experiment tomeasure and connect the chemical shift values of the α-proton of aminoacid residue i, ¹H^(α) _(i), the α-carbon of amino acid residue i,¹³C^(α) _(i), ¹⁵N_(i), and ¹H^(N) _(i). Then, sequential assignments ofthe chemical shift values of ¹H^(ali), ¹³C^(ali), ¹⁵N, and ¹H^(N) areobtained by (i) matching the chemical shift values of the α-proton ofamino acid residue i−1, ¹H^(αi−1), and the α-carbon of amino acidresidue i−1, ¹³C^(α) _(l−1), with the chemical shift values of ¹H^(α)_(l) and ¹³C^(α) _(i), (ii) using the chemical shift values of ¹H^(ali)_(i−1) and ¹³C^(ali) _(i−1) to identify the type of amino acid residuei−1, and (iii) mapping sets of sequentially connected chemical shiftvalues to the amino acid sequence of the polypeptide chain and using thechemical shift values to locate secondary structure elements within thepolypeptide chain.

[0021] Another aspect of the present invention involves a method forobtaining assignments of chemical shift values of ¹H, ¹³C and ¹⁵N of aprotein molecule. The method involves providing a protein sample andconducting four reduced dimensionality (RD) nuclear magnetic resonance(NMR) experiments on the protein sample, where (1) a first experiment isselected from the group consisting of a RD 3D H ^(α/β) C ^(α/β)(CO)NHNNMR experiment, a RD 3D HA,CA,(CO),N,HN NMR experiment, and a RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment for obtaining sequentialcorrelations of chemical shift values; (2) a second experiment isselected from the group consisting of a RD 3D HNNCAHA NMR experiment, aRD 3D H ^(α/β),C ^(α/β),N,HN NMR experiment, and a RD 3D HNN<CO,CA> NMRexperiment for obtaining intraresidue correlations of chemical shiftvalues; (3) a third experiment is a RD 3D H,C,C,H-COSY NMR experimentfor obtaining assignments of sidechain chemical shift values; and (4) afourth experiment is a RD 2D HB,CB,(CG,CD),HD NMR experiment forobtaining assignments of aromatic sidechain chemical shift values.

[0022] The present invention discloses eight new RD TR NMR experimentsand different combinations of those eight experiments as well as threeother RD TR NMR experiments which allows one to obtain sequentialbackbone chemical shift assignments for determining the secondarystructure of a protein molecule and nearly complete assignments ofchemical shift values for a protein molecule including aliphatic andaromatic sidechain spin systems.

[0023] RD NMR spectroscopy is a powerful approach to avoid recording TRNMR data for resonance assignment in the “sampling limited dataacquisition regime.” The set of NMR experiments for HTP structuredetermination as claimed in the present invention allows one toeffectively adapt measurement times to sensitivity requirements. This isof outstanding value in view of HTP protein resonance assignment effortsin the forthcoming era of commercially available cryogenic probes. Inparticular, the rapid determination of a protein's secondary structurecan greatly support fold prediction and thus protein target selectionrequired for structural genomics (Montelione et al., Nature Struc.Biol., 7:982-984 (2000), which is hereby incorporated by reference inits entirety).

[0024] In addition, the present invention which discloses thesensitivity analysis of a suite of TR NMR experiments providing nearlycomplete assignments of chemical shift values of ¹H, ¹³C and ¹⁵N of aprotein molecule is unique and, thus, of general interest for theapplication of TR NMR schemes. The key insights obtained from thisanalysis are (i) that the sensitivity of the individual NMR experimentsconstituting the standard set derived here is comparable or better thanthe 3D HNNCACB NMR experiment, which has so far been routinely employedfor proteins up to about 35 kDa, (Mer et al., J. Biomol. NMR, 17:179-180(2000), which is hereby incorporated by reference in its entirety) and(ii) that data acquisition for most samples of proteins below 20 kDawill be in the undesired sampling limited regime when using conventionalNMR schemes and cryogenic probes. (For 800 MHz systems, such probestoday already offer a sensitivity of 6200:1 for a standard 0.1%ethylbenzene sample (Anderson, “High Q Normal Metal NMR Probe Coils,”42nd Experimental NMR Conference, Orlando, Fla. (2001), which is herebyincorporated by reference in its entirety).) Moreover, the sweep widthsof all indirect dimensions of a multidimensional NMR experiment increasewith increasing magnetic field strength (which implies increasingminimal measurement times). Hence, in view of this concomitant increaseof sensitivity and sweep widths at highest magnetic fields andparticularly considering the anticipated widespread use of cryogenicprobes, a “change in paradigm” in biological NMR spectroscopy isexpected with a new focus on research addressing the caveat of samplinglimitation. This will foreseeably include development and application ofdata processing protocols that allow one to reduce the number of datapoints in the indirect dimensions without concomitantly sacrificingspectral resolution, i.e., linear prediction and maximum entropy methods(Stephenson, Prog. NMR Spectrosc., 20:515-626 (1988), which is herebyincorporated by reference in its entirety), approaches for non-linearsampling (Schmieder et al., J. Biomol. NMR, 4:483-490 (1994); Hoch, etal., NMR Data Processing, Wiley-Liss:New York, (1996), which are herebyincorporated by reference in their entirety), and the recentlyintroduced filter diagonalization method (Wall et al., J. Chem. Phys.,102:8011-8022 (1995); Wall et al., Chem. Phys. Lett., 291:465-470(1998); Hu et al., J. Magn. Reson., 134: 76-87 (1998), which are herebyincorporated by reference in their entirety).

[0025] Considering also random fractional deuteration of proteins forsensitivity enhancement, it is envisioned that the majority of proteinstructure determinations can possibly by accelerated by the applicationof RD NMR spectroscopy. In 2000, there were about eighty 750/800 MHz andthree-hundred 600 MHz spectrometers in operation worldwide, whichrepresent a commercial value of about $350 million (Cross, High FieldNMR: a baseline study., National High Magnetic Field Laboratory,Tallahassee, Fla. (2000), which is hereby incorporated by reference inits entirety). Assuming that about 50% of the instrument time is usedfor NMR structure determination, it is anticipated that the applicationof RD NMR technology promises to greatly impact on the optimized use ofthe large capital invested for NMR-based structural biology.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIGS. 1A-K show the polarization transfer pathways (top) andstick diagrams of the peak pattern observed along ω₁(¹³C) (bottom) forthe RD NMR experiments implemented for the present invention (the 3D H^(α/β) C ^(α/β)(CO)NHN experiment, the 3D HACA(CO)NHN experiment, the 3DHC(C-TOCSY-CO)NHN experiment, the 3D HNNCAHA experiment, the 3D H ^(α/β)C ^(α/β)COHA experiment, the 3D H^(α/β)C^(α/β)NHN experiment, the 3DHNN<CO,CA> experiment, the 3D HCCH-COSY experiment, the 3D HCCH-TOCSYexperiment, the 2D HBCB(CGCD)HD experiment, and the 2D¹H-TOCSY-relayed-HCH-COSY experiment, respectively). The boxes comprisenuclei whose chemical shifts are measured in the common dimension ω₁,and the nuclei which are detected in quadrature in t₁ are marked with anasterisk. Bold solid and hatched boxes indicate intraresidue andsequential connectivities, respectively, and the resulting signalssketched in the stick diagrams are represented accordingly. Those ¹³Cnuclei whose magnetization is used to detect central peaks (Szyperski etal., J. Am. Chem. Soc., 118:8146-8147 (1996), which is herebyincorporated by reference in its entirety), as well as the resultingsubspectrum II shown at the bottom, are highlighted in grey. Themagnetization is frequency labeled with single-quantum coherence of theencircled nuclei during t₂ and detected on the boxed protons. Except forFIG. 1G, the in-phase splittings 2ΔΩ(¹H) are equal to2κ·δΩ(¹H)[γ(¹H)/γ(¹³C)], where κ, δΩ(¹H) and γ(X) denote the scalingfactor applied for ¹H chemical shift evolution (set to 1.0 for thepresent study), the chemical shift difference with respect to theapparent ¹H carrier position, and the gyromagnetic ratio of nucleus X,respectively. In FIG. 1G, the in-phase splittings 2ΔΩ(¹³C^(α)) are equalto 2κ·δΩ(¹³C^(α)), where κ and δΩ(¹³C^(α)) are the scaling factorapplied for ¹³C^(α) chemical shift evolution¹³ (set to 0.5 for thepresent study) and the chemical shift difference with respect to theapparent ¹³C^(α) carrier position, respectively.

[0027]FIG. 2A illustrates the experimental scheme for the 3D H ^(α/β) C^(α/β)(CO)NHN experiment. Rectangular 90° and 180° pulses are indicatedby thin and thick vertical bars, respectively, and phases are indicatedabove the pulses. Where no radio-frequency (r.f.) phase is marked, thepulse is applied along x. The scaling factor κ for ¹H chemical shiftevolution during t₁ is set to 1.0. The high-power 90° pulse lengthswere: 5.9 μs for ¹H, 15.4 μs for ¹³C, and 38 μs for ¹⁵N. Pulses on ¹³Cprior to t₁(¹³C) are applied at high power, and ¹³C decoupling duringt₁(¹H) is achieved using a (90_(x)-180_(y)-90_(x)) composite pulse.Subsequently, the 90° and 180° pulse lengths applied for ¹³C^(α/β) areadjusted to 47.5 μs and 42.5 μs, respectively, to minimize perturbationof ¹³CO spins. The width of the 90° pulse applied on ¹³CO pulse is 52 μsand the corresponding 180° pulses are applied with same power. A SEDUCE180° pulse with a length of 200 μs is used to decouple ¹³CO during t₁and τ₄. The length of the spin-lock purge pulses SL_(x) and SL_(y) are1.2 ms and 0.6 ms, respectively. WALTZ16 is employed to decouple ¹H(r.f. field strength=9.2 kHz) during the heteronuclear magnetizationtransfers as well as to decouple ¹⁵N during acquisition (r.f.=1.78 kHz).The SEDUCE sequence is used for decoupling of ¹³C^(α) during ¹⁵Nevolution period (r.f.=1.0 kHz). The ¹H r.f. carrier is placed at 0 ppmbefore the start of the semi constant time ¹H chemical shift evolutionperiod, and then switched to the water line at 4.78 ppm after the second90° ¹H pulse. Initially, the ¹³C and ¹⁵N r.f. carriers are set to 43 ppmand 120.9 ppm, respectively. The ¹³C carrier is set to 56 ppm during thesecond τ₄/2 delay. The duration and strengths of the pulsed z-fieldgradients (PFGs) are: G1 (1 ms, 24 G/cm); G2 (100 μs, 16 G/cm); G3 (250μs, 29.5 G/cm); G4 (250 μs, 30 G/ cm); G5 (1.5 ms, 20 G/cm); G6 (1.25ms, 30 G/cm); G7 (500 μs, 8 G/cm); G8 (125 μs, 29.5 G/cm). All PFGpulses are of rectangular shape. A recovery delay of at least 100 μsduration is inserted between a PFG pulse and an r.f. pulse. The delaysare: τ₁=800 μs, τ₂=3.1 ms, τ₃=3.6 ms, τ₄=7.2 ms, τ₅=4.4 ms, τ₆=24.8 ms,τ₇=24.8 ms, τ₈=5.5 ms, τ₉=4.6 ms, τ₁₀=1.0 ms. ¹H-frequency labeling isachieved in a semi constant-time fashion with t₁ ^(a) (0)=1.7 ms, t₁^(b) (0)=1 μs, t₁ ^(c) (0)=1.701 ms, Δt₁ ^(a)=33.3 μs, Δt₁ ^(b)=19.3 μs,Δt₁ ^(c)=−14 μs. Hence, the fractional increase of the semiconstant-time period with t₁ equals to λ=1+Δt ₁ ^(c) /Δt ₁ ^(a)=0.58.Phase cycling: φ₁=x; φ₂=x,x,−x,−x; φ₃=x, −x; φ₄=x, −x; φ₅=x; φ6=x, x,−x, −x; φ₇=x; φ₈(receiver)=x, −x, −x, x. The sensitivity enhancementscheme of Kay (Cavanagh et al., Protein NMR Spectroscopy, AcademicPress, San Diego, (1996), which is hereby incorporated by reference inits entirety) is employed, i.e., the sign of G₆ is inverted in concertwith a 180° shift of φ₇. Quadrature detection in t₁(¹³C) and t₂(¹⁵N) isaccomplished by altering the phases φ₂ and φ₅, respectively, accordingto States-TPPI (Cavanagh et al., Protein NMR Spectroscopy, AcademicPress, San Diego, (1996), which is hereby incorporated by reference inits entirety). For acquisition of central peaks derived from ¹³C steadystate magnetization, a second data set with φ₁=−x is collected. The sumand the difference of the two resulting data sets generate subspectra IIand I, respectively, containing the central peaks and peak pairs.

[0028]FIG. 2B illustrates the experimental scheme for the 3D HACA(CO)NHNexperiment. Rectangular 90° and 180° pulses are indicated by thin andthick vertical bars, respectively, and phases are indicated above thepulses. Where no radio-frequency (r.f.) phase is marked, the pulse isapplied along x. The scaling factor κ for ¹H chemical shift evolutionduring t₁ is set to 1.0. The high power 90° pulse lengths were: 5.8 μsfor ¹H and 15.4 μs for ¹³C, and 38 μs for ¹⁵N. Pulses on ¹³C prior tot₁(¹³C) are applied at high power, and ¹³C decoupling during t₁(¹H) isachieved using a (90_(x)-180_(y)-90_(x)) composite pulse. Subsequently,the 90° and 180° pulse lengths of ¹³C^(α) are adjusted to 51.5 μs and 46μs, respectively, to minimize perturbation of the ¹³CO spins. The widthof the 90° pulses applied to ¹³CO pulse is 52 μs and the corresponding180° pulses are applied with same power. A SEDUCE 180° pulse with alength 252 μs is used to decouple ¹³CO during t₁. The length of thespin-lock purge pulses SL_(x) and SL_(y) are 2.5 ms and 1 ms,respectively. WALTZ16 is employed to decouple ¹H (r.f. fieldstrength=9.2 kHz) during the heteronuclear magnetization transfers aswell as to decouple ¹⁵N during acquisition (r.f.=1.78 kHz). The SEDUCEsequence is used for decoupling of ¹³C^(α)during the ¹⁵N chemical shiftevolution period (r.f.=1.0 kHz). The ¹H r.f. carrier is placed at 0 ppmbefore the start of the semi constant time ¹H evolution period, and thenswitched to the water line at 4.78 ppm after the second 90° ¹H pulse.The ¹³C^(α) and ¹⁵N r.f. carriers are set to 56.1 ppm and 120.9 ppm,respectively. The duration and strengths of the pulsed z-field gradients(PFGs) are: G1 (1 ms, 24 G/cm); G2 (100 μs, 16 G/cm); G3 (1 ms, 24G/cm); G4 (250 μs, 30 G/cm); G5 (1.5 ms, 20 G/cm); G6 (1.25 ms, 30G/cm); G7 (500 μs, 8 G/cm); G8 (125 μs, 29.5 G/cm). All PFG pulses areof rectangular shape. A recovery delay of at least 100 μs duration isinserted between a PFG pulse and an r.f. pulse. The delays are: τ₁=1.6ms, τ₂=3.6 ms, τ₃=4.4 ms, τ₄=τ₅=24.8 ms, τ₆=5.5 ms, τ₇=4.6 ms, τ₈=1 ms.¹H-frequency labeling is achieved in a semi constant-time fashion witht₁ ^(a) (0)=1.7 ms, t₁ ^(b) (0)=1 μs, t₁ ^(c) (0)=1.701 ms, Δt₁ ^(a)=60μs, Δt₁ ^(b)=35.4 μs, Δt₁ ^(c)=−24.6 μs. Hence, the fractional increaseof the semi constant-time period with t₁ equals to λ=1+Δt₁ ^(c)/Δt₁^(a)=0.58. Phase cycling: φ₁ =x; φ₂=x, x, −x, −x; φ₃=x, −x; φ₄=x; φ₅=x,x, −x, −x; φ₆=x; φ₇(receiver)=x, −x, −x, x. The sensitivity enhancementscheme of Kay (Cavanagh et al., Protein NMR Spectroscopy, AcademicPress, San Diego, (1996), which is hereby incorporated by reference inits entirety) is employed., i.e., the sign of G6 is inverted in concertwith a 180° shift of Ω₆. Quadrature detection in t₁(¹³C) and t₂(¹⁵N) isaccomplished by altering the phases φ₂ and φ₄, respectively, accordingto States-TPPI (Cavanagh et al., Protein NMR Spectroscopy, AcademicPress, San Diego, (1996), which is hereby incorporated by reference inits entirety). For acquisition of central peaks derived from ¹³C steadystate magnetization, a second data set with φ₁=−x is collected. The sumand the difference of the two resulting data sets generate subspectra IIand I, respectively, containing the central peaks and peak pairs.

[0029]FIG. 2C illustrates the experimental scheme for the 3DHC(C-TOCSY-CO)NHN experiment. Rectangular 90° and 180° pulses areindicated by thin and thick vertical bars, respectively, and phases areindicated above the pulses. Where no radio-frequency (r.f.) phase ismarked, the pulse is applied along x. The scaling factor κ for ¹Hchemical shift evolution during t₁ is set to 1.0. The high power 90°pulse lengths were: 5.8 μs for ¹H and 15.5 μs for ¹³C, and 38 μs for¹⁵N. Pulses on ¹³C prior to t₁(¹³C) are applied at high power, and ¹³Cdecoupling during t₁(¹H) is achieved using a (90_(x)-180_(y)-90_(x))composite pulse. Subsequently, the 90° and 180° pulse lengths appliedfor ¹³C are adjusted to 47.0 μs and 42.5 μs, respectively, to minimizeperturbation of ¹³CO spins. The width of the 90° pulses applied to ¹³COpulse is 52 μs and the corresponding 180μ pulses are applied with samepower. A SEDUCE 180° pulse with a length 200 μs is used to decouple ¹³COduring t₁ and τ₄ period. WALTZ16 is employed to decouple ¹H (r.f. fieldstrength=9.2 kHz) during the heteronuclear magnetization transfers aswell as to decouple ¹⁵N during acquisition (r.f.=1.78 kHz). The SEDUCEsequence is used for decoupling of ¹³C^(α) during the ¹⁵N chemical shiftevolution period (r.f.=1.0 kHz). The ¹H r.f. carrier is placed at 0 ppmbefore the start of the semi constant time ¹H evolution period, and thenswitched to the water line at 4.78 ppm after the second 90° ¹H pulse.The ¹³C and ¹⁵N r.f. carriers are set to 43 ppm and 120.9 ppm,respectively. The lengths of the ¹³C spin-lock purge pulses, SL_(x), are2.5 ms and 1.25 ms, respectively, before and after the carbon—carbontotal correlation spectroscopy (TOCSY) relay. ¹³C isotropic mixing isaccomplished using DIPSI-2 scheme with a r.f. field strength of 8.5 kHz.The duration and strengths of the pulsed z-field gradients (PFGs) are:G1 (2 ms, 30 G/cm); G2 (100 μs, 8 G/cm); G3 (200 μs, 4 G/cm); G4 (2 ms,30 G/cm); G5(1.25 ms, 30 G/cm); G6 (500 μs, 5 G/cm); G7 (125 μs, 29.5G/cm). All PFG pulses are of rectangular shape. A recovery delay of atleast 100 μs duration is inserted between a PFG pulse and an r.f. pulse.The delays are: τ₁=950 μs, τ₂=3.1 ms, τ₃=3.6 ms, τ₄=7.2 ms, τ₅=4.45 ms,τ₆=24.8 ms, τ₇=24.8 ms, τ₈=5.5 ms, τ₉=4.8 ms, τ₁₀=1 ms. ¹H-frequencylabeling is achieved in a semi constant-time fashion with t₁ ^(a) (0)=1.7 ms, t₁ ^(b) (0)=1 μs, t₁ ^(c) (0)=1.701 ms, Δt₁ ^(a)=33.3 μs, Δt₁^(b)=19.3 μs, Δt₁ ^(c)=−14 μs. Hence, the fractional increase of thesemi constant-time period with t₁ equals to λ=1+Δt₁ ^(c) /Δt ₁^(a)=0.58. Phase cycling: φ₁=x; φ₂=x, −x; φ₃=x, x, −x, −x; φ₄=x, −x;φ₅=x, x, −x, −x; φ₆=x, x, −x, −x; φ₇=x; φ₈=4x,4(−x); φ₉=x;φ₁₀(receiver)=x, −x, −x, x. The sensitivity enhancement scheme of Kay(Cavanagh et al., Protein NMR Spectroscopy, Academic Press, San Diego,(1996), which is hereby incorporated by reference in its entirety) isemployed, i.e., the sign of G5 is inverted in concert with a 180° shiftof φ₉. Quadrature detection in t₁(¹³C) and t₂(¹⁵N) is accomplished byaltering the phases φ₂ and φ₇, respectively, according to States-TPPI(Cavanagh et al., Protein NMR Spectroscopy, Academic Press, San Diego,(1996), which is hereby incorporated by reference in its entirety). Foracquisition of central peaks derived from ¹³C steady statemagnetization, a second data set with φ₁=−x is collected. The sum andthe difference of the two resulting data sets generate subspectra II andI, respectively, containing the central peaks and peak pairs.

[0030]FIG. 2D illustrates the experimental scheme for the 3D HNNCAHAexperiment. Rectangular 90° and 180° pulses are indicated by thin andthick vertical bars, respectively, and phases are indicated above thepulses. Where no radio-frequency (r.f.) phase is marked, the pulse isapplied along x. The scaling factor κ for ¹H chemical shift evolutionduring t₁ is set to 1.0. The 90° pulse lengths were: 5.8 μs for ¹H and21.6 μs for ¹³C^(α), and 38 μs for ¹⁵N, where the 90° pulse width for¹³C^(α) is adjusted to generate a null of excitation in the center ofthe CO chemical shift range. The selective 90° ¹H pulse used to flipback the water magnetization is applied for the 1.8 ms with the SEDUCE-1profile. WALTZ16 is employed to decouple ¹H (r.f. field strength=9.2kHz) during the heteronuclear magnetization transfers as well as todecouple of ¹⁵N (r.f. =1.78 kHz) during acquisition. SEDUCE is used fordecoupling of ¹³CO (max. r.f.=3.0 kHz). WURST-2 is used for simultaneousband selective decoupling of ¹³CO and ¹³C^(β) during τ₄ and the ¹H and¹³C chemical shift evolution during t₁. 3.0 kHz sweeps at 176 ppm and 30ppm, respectively, are used for decoupling of ¹³CO and ¹³C^(β) (exceptfor Ser, Thr, Ala). A sweep of 600 Hz is used at 14 ppm to decouple¹³C^(β) of Ala. The ¹H r.f. carrier is placed at the position of thesolvent line at 4.78 ppm for the first three ¹H pulses and the firstWALTZ period, then switched to 0 ppm during the first delay τ₄/2, andsubsequently switched back to the water line at 4.78 ppm during t₁ ^(c).The ¹³C^(α) and ¹⁵N carriers are set to 56.1 ppm and 120.9 ppm,respectively. The duration and strengths of the pulsed z-field gradients(PFGs) are: G1 (500 μs, 8 G/cm); G2 (500 μs, 4 G/cm); G3 (1 ms, 30G/cm); G4 (150 μs, 25 G/cm); G5 (1.25 ms, 30 G/cm); G6 (500 μs, 8 G/cm);G7 (125 μs, 29.57 G/cm). All PFG pulses are of rectangular shape. Arecovery delay of at least 100 μs duration is inserted between a PFGpulse and an r.f. pulse. The delays have the following values: τ₁=4.6ms, τ₂=5.5 ms, τ₃=24 ms, τ₄=2.0 ms, τ₅=500 ms. ¹³ C-frequency labelingis achieved in a semi constant-time fashion with t₁ ^(a) (0)=1.065 ms,t₁ ^(b) (0)=49 μs, t₁ ^(c) (0)=984 μs, Δt₁ ^(a)=65 μs, Δt₁ ^(b)=49 μs,Δt₁ ^(c)=−16 μs. Hence, the fractional increase of the semiconstant-time period with t₁ equals to λ=1+Δt ₁ ^(c) /Δt ₁ ^(a)=0.76.Note that the acquisition starts with the second complex point in t₁,while the first one is obtained by linear prediction. This ensures thata zero first-order phase correction is achieved along ω₁. Phase cycling:φ₁=x, −x; φ₂=x, x, −x, −x; φ₃=x, −x, −x, x; φ₄=x, φ₅=4(x), 4(−x); φ₆=x;φ₇(receiver)=x, −x, −x, x. The sensitivity enhancement scheme of Kay(Cavanagh et al., Protein NMR Spectroscopy, Academic Press, San Diego,(1996), which is hereby incorporated by reference in its entirety) isemployed, i.e., the sign of G5 is inverted in concert with a 180° shiftof φ₆. Quadrature detection in t₁(¹³C) and t₂(¹⁵N) is accomplished byaltering the phases φ₂ and φ₄ according to States-TPPI (Cavanagh et al.,Protein NMR Spectroscopy, Academic Press, San Diego, (1996), which ishereby incorporated by reference in its entirety).

[0031]FIG. 2E illustrates the experimental scheme for the 3D H ^(α/β) C^(α/β)COHA experiment. Rectangular 90° and 180° pulses are indicated bythin and thick vertical bars, respectively, and phases are indicatedabove the pulses. Where no radio-frequency (r.f.) phase is marked, thepulse is applied along x. The scaling factor κ for the ¹H chemical shiftevolution during t₁ is set to 1.0. The high power 90° pulse lengthswere: 5.9 μs for ¹H, 15.4 μs for ¹³C, and 38.2 μs for ¹⁵N. The 90° and180° pulse lengths of ¹³C^(α/β) were adjusted to 47.4 μs and 42.4 μs,respectively, to minimize perturbation of ¹³CO spins. A 200 μs 180°pulse with SEDUCE profile is used to selectively invert ¹³COmagnetization prior to the start of t₁. The 90° and 180° pulses employedfor excitation of ¹³CO and subsequent magnetization transfer back to¹³C^(α) are of rectangular shape and 52 μs and 103 μs duration,respectively. The length of the spin-lock purge pulses SL_(x) and SL_(y)are 2.5 ms and 1 ms, respectively. WALTZ16 is employed to decouple ¹H(r.f. field strength=9.2 kHz) during the heteronuclear magnetizationtransfers, and for decoupling of ¹⁵N (r.f.=1.78 kHz) during acquisition.GARP is used for decoupling of ¹³C^(α) (r.f.=2.5 kHz). The ¹H r.f.carrier is placed at the position of the solvent line at 0 ppm beforethe start of the first semi constant time ¹H evolution period and thenswitched to the water line at 4.78 ppm after the second 90° ¹H pulse.Initially, the ¹³C and ¹⁵N r.f. carriers are set to 43 ppm and 120.9ppm, respectively. The duration and strengths of the pulsed z-fieldgradients (PFGs) are: G1=G2 (100 μs, 15 G/cm); G3 (2 ms, 25 G/cm); G4(100 μs, 10 G/cm); G5 (1 ms, 27 G/cm); G6 (3 ms, 30 G/cm); G7 (1.3 ms,20 G/cm); G8 (130 μs, 14 G/cm). All PFG pulses are of rectangular shape.A recovery delay of at least 100 μs duration is inserted between a PFGpulse and an r.f. pulse. The delays are: τ₁=800 μs, τ₂=2.8 ms, τ₃=3.6ms, τ₄=6.5 ms, τ₅=1.8 ms, τ₆=1 ms, τ₇=2.8 ms, τ₈=3.6 ms. ¹H-frequencylabeling is achieved in a semi constant-time fashion with t₁ ^(a)(0)=1.7 ms, t₁ ^(b) (0)=1 μs, t₁ ^(c) (0)=1.701 ms, Δt₁ ^(a)=33.3 μs,Δt^(b)=19.3 μs, Δt₁ ^(c)=−14 μs. Hence, the fractional increase of thesemi constant-time period with t₁ equals to λ=1+Δt ₁ ^(c) /Δt ₁^(a)=0.58. Phase cycling: φ₁=x; φ_(x)=x, −x; φ₃=x, −x, x, −x; φ₄=x;φ₅(receiver)=x, −x. Quadrature detection in t₁(¹³C) and t₂(¹⁵N) isaccomplished by altering the phases φ₂ and φ₄, respectively, accordingto States-TPPI (Cavanagh et al., Protein NMR Spectroscopy, AcademicPress, San Diego, (1996), which is hereby incorporated by reference inits entirety). For acquisition of central peaks derived from ¹³C steadystate magnetization, a second data set with φ₁=−x is collected. The sumand the difference of the two resulting data sets generate subspectra IIand I, respectively, containing the central peaks and peak pairs(Szyperski et al., J. Am. Chem. Soc., 118:8146-8147 (1996), which ishereby incorporated by reference in its entirety).

[0032]FIG. 2F illustrates the experimental scheme for the 3D H ^(α/β) C^(α/β)NHN experiment. Rectangular 90° and 180° pulses are indicated bythin and thick vertical bars, respectively, and phases are indicatedabove the pulses. Where no radio-frequency (r.f.) phase is marked, thepulse is applied along x. The scaling factor κ for the ¹H chemical shiftevolution during t₁ is set to 1.0. The high power 90° pulse lengthswere: 5.9 μs for ¹H and 15.4 μs for ¹³C, and 38 μs for ¹⁵N. Pulses on¹³C prior to t₁(¹³C) are applied at high power, and ¹³C decouplingduring t₁(¹H) is achieved using a (90_(x)-180_(y)-90_(x)) compositepulse. Subsequently, the 90° and 180° pulse lengths of ¹³C^(α/β) areadjusted to 49 μs and 43.8 μs to minimize perturbation of ¹³CO spins.SEDUCE 180° pulses of 200 μs pulse length are used to decouple ¹³CO.WALTZ16 is employed to decouple ¹H (r.f. field strength=9.2 kHz) duringthe heteronuclear magnetization transfers, as well as to decouple ¹⁵N(r.f.=1.78 kHz). The ¹H carrier is placed at the position of the solventline at 0 ppm during the first semi constant time ¹H evolution period,and then switched to the water line 4.78 ppm after the second 90° ¹Hpulse. The ¹³C and ¹⁵N r.f. carriers are set to 43 ppm and 120.9 ppm,respectively. The duration and strengths of the pulsed z-field gradients(PFGs) are: G1 (1 ms, 24 G/cm); G2 (500 μs, 8 G/cm); G3 (250 μs, 15G/cm); G4 (1 ms, 11 G/cm); G5 (500 μs, 20 G/cm); G6(500 μs, 4 G/cm); G7(125 μs, 29.5 G/cm). All PFG pulses are of rectangular shape. The delaysare: τ₁=800 μs, τ₂=2.8 ms, τ₃=3.3 ms, τ₄=7.2 ms, τ₅=24 ms, τ₆=5.4 ms,τ₇=4.8 ms, τ₈=1 ms. ¹H-frequency labeling is achieved in a semiconstant-time fashion with t₁ ^(a) (0)=1.7 ms, t₁ ^(b) (0)=1 μs, t₁ ^(c)(0)=1.701 ms, Δt₁ ^(a)=33.3 μs, Δt₁ ^(b)=19.3 μs, Δt₁ ^(c)=−14 μs.Hence, the fractional increase of the semi constant-time period with t₁equals to λ=1+Δt ₁ ^(c) /Δt ₁ ^(a)=0.58. Phase cycling: φ₁=x; φ₂=x;φ₃=x,−x; φ₄=x, x, −x, −x; φ₅=x; φ₆(receiver) =x, −x. The sensitivityenhancement scheme of Kay (Cavanagh et al., Protein NMR Spectroscopy,Academic Press, San Diego, (1996), which is hereby incorporated byreference in its entirety) is employed, i.e., the sign of G5 is invertedin concert with a 180° shift of φ₅. Quadrature detection in t₁(¹³C) andt₂(¹⁵N) is accomplished by altering the phases φ₂ and φ₃, respectively,according to States-TPPI. For acquisition of central peaks derived from¹³C steady state magnetization, a second data set with φ₁=−x iscollected. The sum and the difference of the two resulting data setsgenerate subspectra II and I, respectively, containing the central peaksand peak pairs.

[0033]FIG. 2G illustrates the experimental scheme for the 3D HNN<CO,CA>experiment. Rectangular 90° and 180° pulses are indicated by thin andthick vertical bars, respectively, and phases are indicated above thepulses. Where no radio-frequency (r.f.) phase is marked, the pulse isapplied along x. The scaling factor κ for ¹³C^(α) chemical shiftevolution during t₂ is set to 0.5. The high power 90° pulse lengthswere: 5.8 μs for ¹H and 38.5 μs for ¹⁵N. The 90° and 180° pulse lengthsof ¹³C^(α) were adjusted 54 μs and 48.8 μs to minimize perturbation of¹³CO spins. The length of the 90° pulses applied on ¹³CO are 102 μs, andthey possess the shape of a sinc center lobe. The corresponding 180°pulses are applied with same power and shape. The selective ¹H 90° pulseused for flip-back of water magnetization is applied for 1.8 ms with theSEDUCE-1 profile. WALTZ16 is employed to decouple ¹H (r.f. fieldstrength =9.2 kHz) during the heteronuclear magnetization transfers aswell as to decouple ¹⁵N during acquisition (r.f.=1.78 kHz). The SEDUCEsequence is used for decoupling of ¹³C^(α) during ¹⁵N evolution period(r.f.=0.9 kHz). The ¹³C^(α) and ¹⁵N r.f. carriers are set to 176.5 ppmand 120.9 ppm, respectively. The duration and strengths of the pulsedz-field gradients (PFGs) are: G1 (500 μs, 30 G/cm); G2 (500 μs, 5 G/cm);G3 (2 ms, 13 G/cm); G4 (750 μs, 20 G/cm); G5 (200 μs, 5 G/cm); G6 (100μs, 12 G/cm); G7 (1.25 ms, 30 G/cm); G8 (300 μs, 5 G/cm); G9 (200 μs, 10G/cm); G10 (125 μs, 29.5 G/cm). All PFG pulses are of rectangular shape.A recovery delay of at least 100 μs duration is inserted between a PFGpulse and an r.f. pulse. The delays are: τ₁=4.6 ms, τ₂=5.5 ms, τ₃=τ₄=28ms, τ₅=1 ms. Phase cycling: φ₁=x, x, −x, −x; φ₂=x, −x; φ₃=x; φ₄=x;φ₅=4(x), 4(−x); φ₆=x; φ₇(receiver)=x, −x, −x, x. The sensitivityenhancement scheme of Kay (Cavanagh et al., Protein NMR Spectroscopy,Academic Press, San Diego, (1996), which is hereby incorporated byreference in its entirety) is employed, i.e., the sign of G5 is invertedin concert with a 180° shift of φ₆. Quadrature detection in t₁(¹³C) andt₂(¹⁵N) is accomplished by altering the phases φ₂ and φ₄, respectively,according to States-TPPI (Cavanagh et al., Protein NMR Spectroscopy,Academic Press, San Diego, (1996), which is hereby incorporated byreference in its entirety). To shift the apparent ¹³C^(α) carrierposition to 82.65 ppm, i.e., downfield to all ¹³C^(α) resonances, φ₃ isincremented in 60° steps according to TPPI. Note, that the acquisitionwas started with the ninth complex point and the first eight complexpoints along ω₁(¹³CO) were obtained by linear prediction. This ensuresthat a zero first-order phase correction is achieved along ω₁ (Szyperskiet al., J. Magn. Reson., B 108: 197-203 (1995), which is herebyincorporated by reference in its entirety).

[0034]FIG. 2H illustrates the experimental scheme for the 3D HCCH—COSYexperiment. Rectangular 90° and 180° pulses are indicated by thin andthick vertical bars, respectively, and phases are indicated above thepulses. Where no radio-frequency (r.f.) phase is marked, the pulse isapplied along x. The scaling factor κ for ¹H chemical shift evolutionduring t₁ is set to 1.0. The high power 90° pulse lengths were: 5.8 μsfor ¹H and 15.4 μs for ¹³C, and 38 μs for ¹⁵N. The lengths of the ¹Hspin-lock purge pulses are: first SL_(x), 2.8 ms; second SL_(x), 1.7 ms;SL_(y): 4.9 ms. SEDUCE is used for decoupling of ¹³CO during t₁ and t₂(r.f. field strength=1 kHz). WURST is used for decoupling of ¹³C duringacquisition. The ¹H carrier is placed at the position of the solventline at 0 ppm before the start of the first semi constant time ¹Hevolution period, and then switched to the water line at 4.78 ppm afterthe second 90° ¹H pulse. The ¹³C and ¹⁵N r.f. carriers are set to 38 ppmand 120.9 ppm, respectively. The duration and strengths of the pulsedz-field gradients (PFGs) are: G1 (500 μs, 6 G/cm); G2 (500 μs, 7 G/cm);G3 (100 μs, 12 G/cm); G4 (100 μs, 12.5 G/cm); G5 (2 ms, 9 G/cm); G6 (500μs, 5 G/cm); G7 (1.5 ms, 8 G/cm); G8 (400 μs, 6 G/cm). All gradients areapplied along z-axis and are of rectangular shape. All PFG pulses are ofrectangular shape. A recovery delay of at least 100 μs duration isinserted between a PFG pulse and an r.f. pulse. The delays are: τ₁=1.6ms, τ₂=850 μs, τ₃=2.65 ms, τ₄=3.5 ms, τ₅=7 ms, τ₆=1.6 ms, τ₇=3.2 ms.Phase cycling: φ₁=x; φ₂=x, −x; φ₃=x, −x; φ₄=x; φ₅(receiver)=x, −x.Quadrature detection in t₁(¹³C) and t₂(¹³C) is accomplished by alteringthe phases φ₂ and φ₃, respectively, according to States-TPPI (Cavanaghet al., Protein NMR Spectroscopy, Academic Press, San Diego, (1996),which is hereby incorporated by reference in its entirety). Foracquisition of central peaks derived from ¹³C steady statemagnetization, a second data set with φ₁=−x is collected. The sum andthe difference of the two resulting data sets generate subspectra II andI, respectively, containing the central peaks and peak pairs.

[0035]FIG. 2I illustrates the experimental scheme for the 3D HCCH-TOCSYexperiment. Rectangular 90° and 180° pulses are indicated by thin andthick vertical bars, respectively, and phases are indicated above thepulses. Where no radio-frequency (r.f.) phase is marked, the pulse isapplied along x. The scaling factor κ for ¹H chemical shift evolutionduring t₁ is set to 1.0. The high power 90° pulse lengths were: 5.8 μsfor ¹H and 15.4 μs for ¹³C, and 38 μs for ¹⁵N. ¹³C decoupling duringt₁(¹H) is achieved using a (90_(x)-180_(y)-90_(x)) composite pulse. Thelengths of the ¹H spin-lock purge pulses are: first SL_(x), 5.7 ms;second SL_(x), 0.9 ms; SL_(y), 4.3 ms. SEDUCE is used for decoupling of¹³CO during t₁ and t₂ (r.f. field strength=1 kHz), and GARP is employedfor decoupling of ¹³C during acquisition (r.f.=2.5 kHz). The ¹H r.f.carrier is placed at the position of the solvent line at 0 ppm beforethe start of the first semi constant time ¹H evolution period, and thenswitched to the water line at 4.78 ppm after the second 90° ¹H pulse.The ¹³C^(α) and ¹⁵N r.f. carriers are set to 38 ppm and 120.9 ppm,respectively. The length of ¹³C spin-lock purge pulses denoted SL_(x)are of 2 ms duration. ¹³C isotropic mixing is accomplished using theDIPSI-2 scheme (r.f.=8.5 kHz). The duration and strengths of the pulsedz-field gradients (PFGs) are: G1 (100 μs, 16 G/cm); G2 (2 ms, 15 G/cm);G3 (300 μs, 8 G/cm); G4 (500 μs, 30 G/cm); G5 (100 μs, 16 G/cm). All PFGpulses are of rectangular shape. A recovery delay of at least 100 μsduration is inserted between a PFG pulse and an r.f. pulse. The delaysare: τ₁=850 μs, τ₂=3.2 ms. ¹H-frequency labeling in t₁ is achieved in asemi constant-time fashion with t₁ ^(a) (0)=1.7 ms, t₁ ^(b) (0)=1 μs, t₁^(c) (0) =1.701 ms, Δt₁ ^(a)=33.3 μs, Δt₁ ^(b)=19.3 μs, Δt₁ ^(c)=−14 μs.¹³C-frequency labeling in t₂ is achieved in a semi constant-time fashionwith t₂ ^(a) (0)=1120 μs, t₂ ^(b) (0)=62.5 μs, t₂ ^(c) (0)=995 μs, Δt₂^(a)=160 μs, Δt₂ ^(b)=125 μs, Δt₂ ^(c)=−35 μs. These delays ensure thata 90° first-order phase correction is obtained along ω₂(¹³C). Thefractional increases of the semi constant-time period in t₁ equals toλ=1+Δt₂ ^(c)/Δt₂ ^(a)=0.58, and in t₂ equals to λ=1+Δt ₂ ^(c) /Δt ₂^(a)=0.78. Phase cycling: φ₁=x; φ₂=x, −x; φ₃=x; φ₄=2(x), 2(−x);φ₅(receiver)=x,−x. Quadrature detection in t₁(¹³C) and t₂(¹³C) isaccomplished by altering the phases φ₂ and φ₃, respectively, accordingto States-TPPI (Cavanagh et al., Protein NMR Spectroscopy, AcademicPress, San Diego, (1996), which is hereby incorporated by reference inits entirety). For acquisition of central peaks derived from ¹³C steadystate magnetization, a second data set with φ₁=−x is collected. The sumand the difference of the two resulting data sets generate subspectra IIand I, respectively, containing the central peaks and peak pairs.

[0036]FIG. 2J illustrates the experimental scheme for the 2DHBCB(CGCD)HD experiment. Rectangular 90° and 180° pulses are indicatedby thin and thick vertical bars, respectively, and phases are indicatedabove the pulses. Where no radio-frequency (r.f.) phase is marked, thepulse is applied along x. The scaling factor κ for ¹H chemical shiftevolution during t₁ is set to 1.0. The high power 90° pulse lengthswere: 5.8 μs for ¹H and 15.4 μs for ¹³C. The first 180° pulse on ¹³Cprior to t₁(¹³C) is applied at high power. Subsequently, the 90° pulselengths of ¹³CO is adjusted to 66 μs. The 180° ¹³Cr and ¹³C^(aro) pulsesare of gaussian-3 shape and 375 μs duration. WALTZ16is used fordecoupling of ¹H (r.f. field strength=4.5 kHz) during the magnetizationtransfer from ¹³C^(α) to ¹³C^(aro), and GARP is employed to decouple¹³C^(aro) (r.f.=2.5 kHz) during acquisition. The ¹H r.f. carrier isplaced at 0 ppm before the start of the semi constant time ¹H evolutionperiod, and then switched to the water line at 4.78 ppm after the second90° ¹H pulse. The ¹³C r.f. carrier is set to 38 ppm during ω₁(¹³C^(β))and then switched to 131 ppm before the first 90° pulse on ¹³C^(aro)(pulse labeled with φ₄). The duration and strengths of the pulsedz-field gradients (PFGs) are: G1 (500 μs, 2 G/cm); G2 (1 ms, 22 G/cm);G3 (2 ms, 10 G/cm); G4 (1 ms, 5 G/cm); G5 (500 μs, 4 G/cm); G6 (1 ms,−14 G/cm); G7 (500 μs, −2 G/cm). All PFG pulses are of rectangularshape. A recovery delay of at least 100 μs duration is inserted betweena PFG pulse and an r.f. pulse. The delays are: τ₁=1.8 ms, τ₂=8.8 ms,τ₃=71 μs, τ₄=5.4 ms, τ₅=4.2 ms, τ₆=710 μs, τ₇=2.5 ms. ¹H-frequencylabeling is achieved in a semi constant-time fashion with t₁ ^(a)(0)=1.7 ms, t₁ ^(b) (0)=1 μs, t₁ ^(c) (0)=1.701 ms, Δt₁ ^(a)=33.3 μs,Δt₁ ^(b)=19.3 μs, Δt₁ ^(c)=−14 μs. Hence, the fractional increase of thesemi constant-time period with t₁ equals to λ=1+Δt₁ ^(c) /Δt ₁^(a)=0.58. Phase cycling: φ₁=x; φ₂=x; φ₃=x, y, −x, −y; φ₄=4(x), 4(−x);φ₅(receiver)=x, −x, x, −x, −x, x, −x, x. Quadrature detection in t₁(¹³C)is accomplished by altering the phases φ₂ respectively, according toStates-TPPI. For acquisition of central peaks derived from ¹³C steadystate magnetization, a second data set with φ₁=−x is collected. The sumand the difference of the two resulting data sets generate subspectra IIand I, respectively, containing the central peaks and peak pairs.

[0037]FIG. 2K illustrates the experimental scheme for the 2D¹H-TOCSY-relayed-HCH-COSY experiment. Rectangular 90° and 180° pulsesare indicated by thin and thick vertical bars, respectively, and phasesare indicated above the pulses. Where no radio-frequency (r.f.) phase ismarked, the pulse is applied along x. The high-power 90° pulse lengthswere: 5.9 μs for ¹H and 15.4 μs for ¹³C. The ¹H r.f. carrier is placedat the position of the solvent line at 4.78 ppm, and the ¹³C carrier isset to 131 ppm. GARP is used for ¹³C decoupling during acquisition (r.f.field strength=2.5 kHz), and ¹H isotropic mixing is accomplished usingthe DIPSI-2 scheme (r.f.=16 kHz). The duration and strengths of thepulsed z-field gradients (PFGs) are: G1 (1 ms, −10 G/cm); G2 (500 μs, 6G/cm); G3 (500 μs, 7.5 G/cm); G4 (1 ms, 22 G/cm). All PFG pulses are ofrectangular shape. A recovery delay of at least 100 μs duration isinserted between a PFG pulse and an r.f. pulse. The delays are: τ₁=3.0ms, τ₂=15.38 ms. Phase cycling: φ₁=x, −x; φ₂=x, x, y, y, −x, −x, −y, −y;φ₃=4(x), 4(y), 4(−x), 4(−y); φ₄=x, x, −x, −x; φ₅(receiver)=x, −x, x, −x,−x, x, −x, x. Quadrature detection in t₁(¹³C) is accomplished byaltering the phase φ₁ according to States-TPPI.

[0038]FIG. 3 illustrates the polypeptide chemical shifts correlated bythe various spectra constituting the “standard set” of TR NMRexperiments identified for efficient HTP resonance assignment ofproteins. The nuclei for which the chemical shifts are obtained from agiven experiment are boxed and labeled accordingly.

[0039] FIGS. 4A-F illustrate the sequential connectivities in RD NMRspectra. FIGS. 4A-C show the cross sections along ω¹(¹³C) taken from thesubspectra I, which exhibit peak pairs arising from ¹H chemical shiftevolution, of 3D HACA(CO)NHN (FIG. 4A), 3D H ^(α/β) C ^(α/β)(CO)NHN(FIG. 4B) and 3D HC(C-TOCSY-CO)NHN (FIG. 4C) (21 ms mixing time). Thein-phase splittings encoding the ¹H^(α) (FIG. 4A), ¹H^(β) (FIG. 4B) and¹H^(γ) (FIG. 4C) chemical shifts of Gln 26 are indicated. FIGS. 4D-Fshow the corresponding cross sections taken from subspectra II, whichexhibit central peaks. The signals arising from ¹³C^(α) (FIG. 4A),¹³C^(β) (FIG. 4B) and ¹³C^(γ) (FIG. 4C) of Gln 26 are indicated.Chemical shifts are given relative to2,2-dimethyl-2-silapentane-5-sulfonate (DSS).

[0040]FIG. 5 illustrates the sensitivity of TR NMR experiments relativeto peak pair detection in 3D HACA(CO)NHN (left-most bar). As indicatedon the top of the figure, the experiments are grouped according toproviding interresidue (“inter”), intraresidue (“intra”), aliphatic sidechain (“ali”) and aromatic side chain (“aro”) connectivities. For RD NMRexperiments, the sensitivity of peak pair (black bars) and central peak(grey bars) detection was analyzed separately. The yield of peakdetection (in percent) is indicated on the top of the bars. Note thatonly those peak categories encoding the prime information to be obtainedfrom a given spectrum, i.e., intraresidual connectivities in HNNCAHA(FIG. 1D), H ^(α/β) C ^(α/β)COHA (FIG. 1E), H ^(α/β) C ^(α/β)NHN (FIG.1F) and HNNCACB, correlation peaks in HCCH-COSY and relay connectivitiesin HCCH TOCSY, and only peaks exhibiting a S/N ratio larger than 3 wereconsidered for this plot. For well-resolved RD peak pairs the averagedS/N ratio of the two individual peaks is given. In cases where only oneof the two peaks is well resolved, only the value for the resolved peakwas considered. As an example, the insert shows the S/N distributionsobtained for the intraresidue peak pairs detected in 3D HNNCAHA. Blackand grey bars correspond to spectra acquired with and without adiabatic¹³C^(β)-decoupling, respectively (Abragam, Principles of NuclearMagnetism, Clarendon Press:Oxford (1986); Ernst et al., Principles ofNuclear Magnetic Resonance in One and Two Dimensions, ClarendonPress:Oxford (1987), which are hereby incorporated by reference in theirentirety).

[0041] FIGS. 6A-C illustrate the intraresidue connections in RD NMRspectra: cross sections along ω₁(¹³C) taken from 3D HNNCAHA (FIG. 6A),subspectrum I of 3D H ^(α/β) C ^(α/β)COHA (FIG. 6B) and subspectrum I of3D H ^(α/β) C ^(α/β)NHN (FIG. 6C). The in-phase splittings encoding the¹H^(α) of Glu 24 and Asn 23 (FIG. 6A) and ¹H^(β) of Glu 24 (FIG. 6B) areindicated. Chemical shifts are given relative to DSS.

[0042]FIG. 7 is the schematic presentation of the RD NMR-based HTPresonance assignment strategy using the “standard set” of experimentsidentified in the framework of the present study. The central role of 3DH ^(α/β) C ^(α/β)(CO)NHN is shown for creating sequential connectivitiesvia (i) ¹³C^(α) and ¹H^(α) shift measurements (HNNCAHA; FIG. 8), via(ii) ¹³C^(α) and ¹³C^(β) shift measurements (HNNCACB), and via (iii)¹³C═O shift measurements (H ^(α/β) C ^(α/β)COHA/HNNCAHA and HNN<CO,CA>;FIG. 9). This key role is further evidenced when employing 3D H ^(α/β) C^(α/β)(CO)NHN also for assigning aliphatic (HCCH-COSY/TOCSY; FIGS. 9 and10) and aromatic side chains (HBCB(CGCD)HD and ¹H-TOCSY-relayedHCH-COSY; FIG. 12). Black double-headed arrows indicate connectivitieswhich are established based on matching of peak patterns along ω₁(¹³C)of the spectra, and grey arrows indicate that the combined use of thetwo spectra connected by the arrow requires the conversion of in-phasesplittings into chemical shifts. Each box shows the peak patternsexpected along ω₁, and the chemical shifts that are measured in theother dimensions are given above the corresponding boxes. Two crosssections are sketched for RD NMR experiments which yield two subspectralabeled with I and II, which comprise peak pairs and central peaks,respectively.

[0043]FIG. 8 shows the sequential resonance assignment from 3D H ^(α/β)C ^(α/β)(CO)NHN/3D HNNCAHA. Contour plot of [ω₁(¹³C), ω₃(¹H_(N))]-stripstaken from subspectrum I (strips labeled with AI) and subspectrum II(strips labeled with AII) of 3D H ^(α/β) C ^(α/β)(CO)NHN, and from 3DHNNCAHA (strips labeled with B) are shown. The strips were taken at the¹⁵N chemical shifts (indicated at the top) of residues 51 to 55 and arecentered about their ¹H^(N) chemical shift. The sequence-specificresonance assignments of the amide chemical shifts are given at the topof each strip and are referred to as i. Ω(¹H^(α/β) _(i−1)) andΩ(¹³C^(α/β) _(l−1)) obtained from 3D H ^(α/β) C ^(α/β)(CO)NHN are givenin the strips AI and AII of residue i. Corresponding peak pairs in AIand central peaks in AII are connected by dashed lines, and sequentialconnectivities are indicated by solid lines for both peak pairs andcentral peaks. Dashed and solid contour lines represent negative andpositive peaks, respectively, and sequential connectivities establishedvia the central peaks and via the peak pairs are indicated by solid anddotted lines, respectively. Note, that the near-degeneracy of ¹³C^(α)chemical shifts in the polypeptide segment Asn 52-Asp 53-Ala 54 isneatly resolved by the measurement of ¹H^(α) chemical shifts encoded inthe in-phase splittings of the peak pairs. Chemical shifts are relativeto DSS.

[0044] FIGS. 9A-B show the sequential resonance assignment based on 3D H^(α/β) C ^(α/β)(CO)NHN/3D H ^(α/β) C ^(α/β)COHA combined with 3DHNN<CA,CO> (FIG. 7). The amino acid residue on which the NMR signal wasdetected is indicated at the bottom of the strips. FIG. 9A shows thematching of ω₁(¹³C^(α/β)) peak patterns in 3D H ^(α/β) C ^(α/β)COHA(strips labeled with “a”) and 3D H ^(α/β) C ^(α/β)(CO)NHN (“b”) yieldsputative intraresidue ¹H^(α/β)/¹³ C^(α/β)—¹³ C═O correlations: on thestrips taken from 3D H ^(α/β) C ^(α/β)COHA the ¹³C═O chemical shift isindicated. 3D HNN<CA,CO> yields the sequential ¹³C═O—¹³C^(α)correlations (FIG. 9B): the carbonyl chemical shifts have to match thoseshown in FIG. 9A, and are indicated on the left of the figure. Protonand carbon chemical shifts are given in ppm and are relative to DSS.

[0045] FIGS. 10A-C shows the assignment of aliphatic spin systems using3D H ^(α/) ^(β) C ^(α/β)(CO)NHN/3D HCCH-COSY exemplified for Lys 4.Cross sections taken along ω₁(¹³C) from 3D H ^(α/β) C ^(α/β)(CO)NHN(FIG. 10A) and subspectrum I of 3D HCCH-COSY (FIG. 10B) are shown. Thesignals in 3D H ^(α/β) C ^(α/β)(CO)NHN were detected on the backboneamid proton of the succeeding residue Phe 5 (the ¹⁵N and ¹H^(N) chemicalshifts are indicated on the right). The cross sections taken fromHCCH-COSY exhibit signals which were detected on ¹H^(α), ¹H^(β), ¹H^(γ)and ¹H^(δ) of Lys 4, respectively (from the bottom to the top). Thein-phase splittings encode the ¹H^(β), ¹H^(γ), ¹H^(δ) and ¹H^(ε) 0chemical shifts and serve to obtain the desired correlations asindicated by dashed vertical lines. Note that the peak signs varybecause of aliasing along ω₂(¹³C). In FIG. 10C, a ω₁(¹³C) cross sectionfrom 3D HCCH-TOCSY is shown. The signal was detected on ¹H^(γ) of Lys 4,and the crucial ^(α)CH—^(γ)CH relay connectivity is indicated (see alsoFIG. 9). Proton and carbon chemical shifts are relative to DSS.

[0046] FIGS. 11A-C show the assignment of aliphatic side chainsexemplified for Lys 4 (see also FIG. 12). Pairs of cross sections takenfrom 3D HCCH-COSY and TOCSY are shown. These exhibit signals detected on¹H^(α) (FIG. 11A), ¹H^(β) (FIG. 11B) and ¹H^(γ) (FIG. 11C) of Lys 4,respectively. The crucial ^(α)CH—^(γ)CH relay connectivities, whichresolve potential overlap in HCCH-COSY, are indicated with verticallines. Note that the peak signs vary because of aliasing along ω₂(¹³C).The assignment of the peak pairs is shown in FIG. 10.

[0047] FIGS. 12A-C show the assignment of aromatic side chainsexemplified for His(−4) and His 18, and Tyr 14. A composite plot of[ω₁(¹³C),ω₃(¹H^(N))]-strips taken from 3D H ^(α/β) C ^(α/β)(CO)NHNcomprising the ω₁(¹³C) peaks of all aromatic side chains in thepolypeptide segment (−5)-58 of Z-domain, the 2D HBCB(CGCD)HD spectrum(FIG. 12B) as well as a spectral region taken from 2D ¹H-TOCSY-relayedHCH-COSY (FIG. 12C) are shown. The entire 2D ¹H-TOCSY-relayed HCH-COSYspectrum, which also contains cross peaks arising from ^(ε)CH of thehistidinyl residues, is shown in the upper right of the figure.Correlations belonging to His(−4), His 18, and Tyr 14 are connected withlong-dashed, dashed and grey solid lines, respectively. In FIG. 12C,peaks arising from ^(α)CH moieties (which are not required forconnecting the aromatic spin systems) are labeled with an asterisk.

DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention discloses eight new RD TR NMR experimentsand different combinations of those eight experiments as well as threeother RD TR NMR experiments which allows one to obtain sequentialbackbone chemical shift assignments for determining the secondarystructure of a protein molecule and nearly complete assignments ofchemical shift values for a protein molecule including aliphatic andaromatic sidechain spin systems. FIG. 1 provides a survey of (i) thenames, (ii) the magnetization transfer pathways and (iii) the peakpatterns observed in the projected dimension of specific embodiments ofthe 8 new RD NMR experiments disclosed by the present invention as wellas 3 other RD NMR experiments that have previously been published. Thegroup comprising the first three experiments are designed to yield“sequential” connectivities via one-bond scalar couplings: 3D H ^(α/β) C^(α/β)(CO)NHN (FIG. 1A; Szyperski et al., J. Magn. Reson., B 105:188-191 (1994), which is hereby incorporated by reference in itsentirety), 3D HACA(CO)NHN (FIG. 1B), and 3D HC(C-TOCSY-CO)NHN (FIG. 1C).The following three experiments provide “intraresidual” connectivitiesvia one-bond scalar couplings: 3D HNNCAHA (FIG. 1D; Szyperski et al., J.Biomol. NMR, 11:387-405 (1998), which is hereby incorporated byreference in its entirety), 3D H ^(α/β) C ^(α/β)COHA (FIG. 1E), and 3D H^(α/β) C ^(α/β)NHN (FIG. 1F). 3D HNN<CO,CA> (FIG. 1G; Szyperski et al.,J. Magn. Reson., B 108: 197-203 (1995); Szyperski et al., J. Am. Chem.Soc., 118:8146-8147 (1996), which are hereby incorporated by referencein their entirety) offers both intraresidual ¹H^(N)—¹³C^(α) andsequential ¹H^(N)—¹³C′ connectivities. Although 3D HNNCAHA (FIG. 1D), 3DH ^(α/β) C ^(α/β)NHN (FIG. 1F) and 3D HNN<CO,CA> (FIG. 1G) also providesequential connectivities via two-bond ¹³C^(α) _(i−1)—¹⁵N_(l) scalarcouplings, those are usually smaller than the one-bond couplings(Cavanagh et al., Protein NMR Spectroscopy, Academic Press, San Diego,(1996), which is hereby incorporated by reference in its entirety), andobtaining complete backbone resonance assignments critically depends onexperiments designed to provide sequential connectivities via one-bondcouplings (FIGS. 1D-F). 3D HCCH-COSY (FIG. 1H) and 3D HCCH-TOCSY (FIG.1I) allow one to obtain assignments for the “aliphatic” side chain spinsystems, while 2D HBCB(CDCG)HD (FIG. 1J) and 2D ¹H-TOCSY-relayedHCH-COSY (FIG. 1K) provide the corresponding information for the“aromatic” spin systems.

[0049] The RD NMR experiments are grouped accordingly in Table 1, whichlists for each experiment (i) the nuclei for which the chemical shiftsare measured, (ii) if and how the central peaks are acquired and (iii)additional notable technical features. State-of-the art implementations(Cavanagh et al., Protein NMR Spectroscopy, Academic Press, San Diego,(1996); Kay, J. Am. Chem. Soc., 115:2055-2057 (1993); Grzesiek et al.,J. Magn. Reson., 99:201-207 (1992); Montelione et al., J. Am. Chem.Soc., 114:10974-10975 (1992); Boucher et al., J. Biomol. NMR, 2:631-637(1992); Yamazaki et al., J. Am. Chem. Soc., 115:11054-11055 (1993);Zerbe et al., J. Biomol. NMR, 7:99-106 (1996); Grzesiek et al., J.Biomol. NMR, 3:185-204 (1993), which are hereby incorporated byreference in their entirety) making use of pulsed field z-gradients forcoherence selection and/or rejection, and sensitivity enhancement(Cavanagh et al., Protein NMR Spectroscopy, Academic Press, San Diego,(1996), which is hereby incorporated by reference in its entirety) werechosen, which allow executing these experiments with a single transientper acquired free induction decay (FID). Semi (Grzesiek et al., J.Biomol. NMR, 3:185-204 (1993), which is hereby incorporated by referencein its entirety) constant-time (Cavanagh et al., Protein NMRSpectroscopy, Academic Press, San Diego, (1996), which is herebyincorporated by reference in its entirety) chemical shiftfrequency-labeling modules were used throughout in the indirectdimensions in order to minimize losses arising from transverse nuclearspin relaxation. FIGS. 2A-2K provide comprehensive descriptions of theRD NMR r.f. pulse sequences used in the 11 RD NMR experiments includingeight previously unpublished RD NMR r.f. pulse schemes. TABLE 1 ReducedDimensionality NMR Experiments for HTP Resonance Assignment ExperimentNuclei for which the chemical shifts are Acquisition of (see FIG. 1)correlated^(a,b) central peaks^(c) 3D spectra for sequential backboneconnectivities: (A) H ^(α/β) C ^(α/β)(CO)NHN ¹H^(β) _(i-1) ¹³C^(β)_(i-l), ¹H^(α) _(i-1), ¹³C^(α) _(i-1), ¹⁵N_(i), ¹H^(N) _(i) ¹³C (B)HACA(CO)NHN ¹H^(α) _(i-1), ¹³C^(α) _(i-1), ¹⁵N_(l), ¹H^(N) _(l) ¹³C (C)HC(C-TOCSY- CO)NHN ¹H^(a1i) _(i-1), ¹³C^(a1i) _(i-1), ¹⁵N_(l), ¹H^(N)_(l) ¹³C 3D spectra for intraresidual backbone connectivities: (D)HNNCAHA ^(b,d) ¹H^(α) _(l), ¹³C^(α) _(l), ¹⁵N_(l), ¹H^(N) _(l) INEPT (E)H ^(α/β) C ^(α/β)COHA ¹H^(β) _(l), ¹³C^(β) _(l), ¹H^(α) _(l), ¹³C^(α)_(l), ¹³C = O_(l) ¹³C (F) H ^(α/β) C ^(α/β)NH ¹H^(β) _(l), ¹³C^(β) _(l),¹H^(α) _(l), ¹³C^(α) _(l), ¹⁵N_(l), ¹H^(N) _(l) ¹³C 3D spectrum forintra- and sequential backbone connectivities: (G) HNN<CO,CA>^(b) ¹³C =O_(i-1), ¹³C^(α) _(l), ¹⁵N_(l), INEPT ¹H^(N) _(l) 3D spectra forassignment of aliphatic resonances:^(e) (H) HCCH-COSY ¹H_(m), ¹³C_(m),¹H_(n), ¹³C_(n) ¹³C (I) HCCH-TOCSY ¹H_(m), ¹³C_(m), ¹H_(n), ¹³C_(n),¹H_(p), ¹³C_(p) ¹³C 2D spectra for assignment of aromaticresonances:^(e) (J) HBCB(CGCD)HD ¹H^(β), ¹³C^(β), ¹H^(δ) ¹³C (K)¹H-TOCSY-HCH-COSY ¹H_(m), ¹³C_(m), ¹H_(n), none^(f) #“¹³C”).

The 3D HA,CA,(CO),N,HN Experiment

[0050] The present invention relates to a method of conducting a reduceddimensionality (RD) three-dimensional (3D) HA,CA,(CO),N,HN nuclearmagnetic resonance (NMR) experiment by measuring the chemical shiftvalues for the following nuclei of a protein molecule having twoconsecutive amino acid residues, i−1 and i: (1) an α-proton of aminoacid residue i−1, ¹H^(α) _(i−1); (2) an α-carbon of amino acid residuei−1, ¹³C^(α) _(l−1); (3) a polypeptide backbone amide nitrogen of aminoacid residue i, ¹⁵N_(l); and (4) a polypeptide backbone amide proton ofamino acid residue i, ¹H^(N) _(l). The method involves providing aprotein sample and applying radiofrequency pulses to the protein samplewhich effect a nuclear spin polarization transfer where the chemicalshift evolutions of ¹H^(α) _(i−1) and ¹³C^(α) _(l−1) of amino acidresidue i−1 are connected to the chemical shift evolutions of ¹⁵N_(i)and ¹H^(N) _(i) of amino acid residue i, under conditions effective (1)to generate NMR signals encoding the chemical shift values of ¹³C^(α)_(l−1) and ¹⁵N_(i) in a phase sensitive manner in two indirect timedomain dimensions, t₁(¹³C^(α)) and t₂(¹⁵N), respectively, and thechemical shift value of ¹H^(N) _(l) in a direct time domain dimension,t₃(¹H^(N)), and (2) to cosine modulate the ¹³C^(α) _(l−1) chemical shiftevolution in t₁(¹³C^(α)) with the chemical shift evolution of ¹H^(α)_(l−1). Then, the NMR signals are processed to generate a 3D NMRspectrum with a primary peak pair derived from the cosine modulating,where (1) the chemical shift values of ¹⁵N_(i) and ¹H^(N) _(l) aremeasured in two frequency domain dimensions, ω₂(¹⁵N) and ω₃(¹H^(N)),respectively, and (2) the chemical shift values of ¹H^(α) _(i−1) and¹³C^(α) _(i−1) are measured in a frequency domain dimension,ω₁(¹³C^(α)), by the frequency difference between the two peaks formingthe primary peak pair and the frequency at the center of the two peaks,respectively.

[0051] In addition, the method of conducting a RD 3D HA,CA,(CO),N,HN NMRexperiment can involve applying radiofrequency pulses under conditionseffective (1) to generate an additional NMR signal encoding the chemicalshift values of ¹³C^(α) _(l−1) and ¹⁵N_(l) in a phase sensitive mannerin t₁(¹³C^(α)) and t₂(¹⁵N) and the chemical shift value of ¹H^(N) _(i)in t₃(¹H^(N)), and (2) to avoid cosine modulating the ¹³C^(α) _(i−1)chemical shift evolution in t₁(¹³C^(α)) with the chemical shiftevolution of ¹H^(α) _(i−1) for the additional NMR signal. Then, the NMRsignals and the additional NMR signal are processed to generate a 3D NMRspectrum with an additional peak located centrally between two peaksforming the primary peak pair which measures the chemical shift value of¹³C^(α) _(i−1) along ω₁(¹³C^(α)). That additional peak can be derivedfrom ¹³C^(α) nuclear spin polarization. One specific embodiment (3DHACA(CO)NHN) of this method is illustrated in FIG. 1B, where theapplying radiofrequency pulses effects a nuclear spin polarizationtransfer where a radiofrequency pulse is used to create transverse¹H^(α) _(l−1) magnetization, which is transferred to ¹³C^(α) _(i−1), to¹⁵N_(l), and to ¹H^(N) _(l), to generate the NMR signal. Anotherspecific embodiment of this method involves applying radiofrequencypulses by (1) applying a first set of radiofrequency pulses according tothe scheme shown in FIG. 2B to generate a first NMR signal, and (2)applying a second set of radiofrequency pulses according to the schemeshown in FIG. 2B, where phase φ₁ of the first ¹H pulse is altered by180° to generate a second NMR signal. Then, prior to the processing, thefirst NMR signal and the second NMR signal are added and subtractedwhereby the NMR signals are processed to generate a first NMRsubspectrum derived from the subtracting which contains the primary peakpair and a second NMR subspectrum derived from the adding which containsthe additional peak located centrally between the two peaks forming theprimary peak pair.

[0052] In addition, the method of conducting a RD 3D HA,CA,(CO),N,HN NMRexperiment can involve applying radiofrequency pulses under conditionseffective to additionally cosine modulate the ¹³C^(α) _(i−1) chemicalshift evolution in t₁(¹³C^(α)) with the chemical shift evolution of apolypeptide backbone carbonyl carbon of amino acid residue i−1,¹³C′_(i−1). Then, the NMR signals are processed to generate a 3D NMRspectrum with two secondary peak pairs where (1) each of the secondarypeak pairs is derived from a different one of the peaks of the primarypeak pair, and (2) the chemical shift value of ¹³C′_(i−1) is measuredalong ω₁(¹³C^(α)) by the frequency difference between the two peaksforming one of the secondary peak pairs. This method can further involveapplying radiofrequency pulses under conditions effective (1) togenerate an additional NMR signal encoding the chemical shift values of¹³C^(α) _(l−1) and ¹⁵N_(i) in a phase sensitive manner in t₁(¹³C^(α))and t₂(¹⁵N) and the chemical shift value of ¹H^(N) _(i) in t₃(¹H^(N)),(2) to cosine modulate the ¹³C^(α) _(l−1) chemical shift evolution int₁(¹³C^(α)) with the chemical shift evolution of ¹³C′_(i−1), and (3) toavoid cosine modulating the ¹³C^(α) _(l−1) chemical shift evolution int₁(¹³C^(α)) with the chemical shift evolution of ¹H^(α) _(i−1). Then,the NMR signals and the additional NMR signal are processed to generatea 3D NMR spectrum with an additional secondary peak pair located betweenthe two secondary peak pairs which measures the chemical shift values of¹³C′_(l−1) and ¹³C^(α) _(l−1) along ω₁(¹³C^(α)), by the frequencydifference between the two peaks forming the additional secondary peakpair and the frequency at the center of the two peaks, respectively.That additional secondary peak pair can be derived from ¹³C^(α)nuclearspin polarization. One specific embodiment (3D HACA(CO)NHN) of thismethod is illustrated in FIG. 1B, where the applying radiofrequencypulses effects a nuclear spin polarization transfer where aradiofrequency pulse is used to create transverse ¹H^(α) _(i−1)magnetization, which is transferred to ¹³C^(α) _(l−1), to ¹⁵N_(l), andto ¹H^(N) _(l), to generate the NMR signal. Another specific embodimentof this method involves applying radiofrequency pulses by (1) applying afirst set of radiofrequency pulses according to the scheme shown in FIG.2B to generate a first NMR signal, and (2) applying a second set ofradiofrequency pulses according to the scheme shown in FIG. 2B, wherephase φ₁ of the first ¹H pulse is altered by 180° to generate a secondNMR signal. Then, prior to the processing, the first NMR signal and thesecond NMR signal are added and subtracted whereby the NMR signals areprocessed to generate a first NMR subspectrum derived from thesubtracting which contains the two secondary peak pairs and a second NMRsubspectrum derived from the adding which contains the additional peaklocated centrally between the primary peak pair.

[0053] In an alternate embodiment, the RD 3D HA,CA,(CO),N,HN NMRexperiment can be modified to a RD 2D HA,CA,(CO,N),HN NMR experimentwhich involves applying radiofrequency pulses so that the chemical shiftevolution of ¹⁵N_(l) does not occur. Then, the NMR signals are processedto generate a two dimensional (2D) NMR spectrum with a peak pair where(1) the chemical shift value of ¹H^(N) _(i) is measured in a frequencydomain dimension, ω₂(¹H^(N)), and (2) the chemical shift values of¹H^(α) _(i−1) and ¹³C^(α) _(i−1) are measured in a frequency domaindimension, ω₁(¹³C^(α)), by the frequency difference between the twopeaks forming the primary peak pair and the frequency at the center ofthe two peaks, respectively.

[0054] In an alternate embodiment, the RD 3D HA,CA,(CO),N,UN NMRexperiment can be modified to a RD 4D HA,CA,CO,N,HN NMR experiment whichinvolves applying radiofrequency pulses so that the chemical shiftevolution of a polypeptide backbone carbonyl carbon of amino acidresidue i−1, ¹³C′_(i−1), occurs under conditions effective to generateNMR signals encoding the chemical shift value of ¹³C′_(i−1) in a phasesensitive manner in an indirect time domain dimension, t₄(¹³C′). Then,the NMR signals are processed to generate a four dimensional (4D) NMRspectrum with a peak pair where (1) the chemical shift values of¹⁵N_(l), ¹H^(N) _(i) and ¹³C′_(i−1) are measured in three frequencydomain dimensions, ω₂(¹⁵N), ω₃(¹H^(N)), and ω₄(¹³C′), respectively, and(2) the chemical shift values of ¹H^(α) _(l−1) and ¹³C^(α) _(l−1) aremeasured in a frequency domain dimension, ω₁(¹³C^(α)), by the frequencydifference between the two peaks forming the peak pair and the frequencyat the center of the two peaks, respectively.

The 3D H,C,(C-TOCSY-CO),N,HN Experiment

[0055] The present invention also relates to a method of conducting areduced dimensionality (RD) three-dimensional (3D) H,C,(C-TOCSY-CO),N,HNnuclear magnetic resonance (NMR) experiment by measuring the chemicalshift values for the following nuclei of a protein molecule having twoconsecutive amino acid residues, i−1 and i: (1) aliphatic protons ofamino acid residue i−1, ¹H^(ali) _(i−1); (2) aliphatic carbons of aminoacid residue i−1, ¹³C^(ali) _(i−1); (3) a polypeptide backbone amidenitrogen of amino acid residue i, ¹⁵N_(i); and (4) a polypeptidebackbone amide proton of amino acid residue i, ¹H^(N) _(i). The methodinvolves providing a protein sample and applying radiofrequency pulsesto the protein sample which effect a nuclear spin polarization transferwhere the chemical shift evolutions of ¹H^(ali) _(l−1) and ¹³C^(ali)_(l−1) of amino acid residue i−1 are connected to the chemical shiftevolutions of ¹⁵N_(l) and ¹H^(N) _(i) of amino acid residue i, underconditions effective (1) to generate a NMR signal encoding the chemicalshifts of ¹³C^(ali) _(l−1) and ¹⁵N_(l) in a phase sensitive manner intwo indirect time domain dimensions, t₁(¹³C^(ali)) and t₂(¹⁵N),respectively, and the chemical shift of ¹H^(N) _(i) in a direct timedomain dimension, t₃(¹H^(N)), and (2) to cosine modulate the chemicalshift evolutions of ¹³C^(ali) _(i−1) in t₁(¹³C^(ali)) with the chemicalshift evolutions of ¹H^(ali) _(i−1). Then, the NMR signals are processedto generate a 3D NMR spectrum with peak pairs derived from the cosinemodulating where (1) the chemical shift values of ¹⁵N_(l) and ¹H^(N)_(l) are measured in two frequency domain dimensions, ω₂(¹⁵N) andω₃(¹H^(N)), respectively, and (2) the chemical shift values of ¹H^(ali)_(i‘)and ¹³C^(ali) _(i−1) are measured in a frequency domain dimension,ω₁(¹³C^(ali)), by the frequency differences between the two peaksforming the peak pairs and the frequencies at the center of the twopeaks, respectively.

[0056] In addition, the method of conducting a RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment can involve applying radiofrequencypulses under conditions effective (1) to generate an additional NMRsignal encoding the chemical shift values of ¹³C^(ali) _(i−1) and¹⁵N_(i) in a phase sensitive manner in t₁(¹³C^(ali)) and t₂(¹⁵N) and thechemical shift value of ¹H^(N) _(l) in t₃(¹H^(N)), and (2) to avoidcosine modulating the chemical shift evolutions of ¹³C^(ali) _(l−1) int₁(¹³C^(ali)) with the chemical shift evolution of ¹H^(α) _(l−1) for theadditional NMR signal. Then, the NMR signals and the additional NMRsignal are processed to generate a 3D NMR spectrum with additional peakslocated centrally between two peaks forming the peak pairs which measurethe chemical shift values of ¹³C^(ali) _(i−1) along ω₁(¹³C^(ali)). Thoseadditional peaks can be derived from ¹³C^(ali) nuclear spinpolarization. One specific embodiment (3D HC-(C-TOCSY-CO)NHN) of thismethod is illustrated in FIG. 1C, where the applying radiofrequencypulses effects a nuclear spin polarization transfer, where aradiofrequency pulse is used to create transverse ¹H^(ali) _(i−1)magnetization, and ¹H^(ali) _(i−l) magnetization is transferred to¹³C^(ali) _(i−1), to ¹³C^(α) _(i−1), to ¹³C′_(i−1), to ¹⁵N_(i), and to¹H^(N) _(i), where the NMR signal is detected. Another specificembodiment of this method involves applying radiofrequency pulses by (1)applying a first set of radiofrequency pulses according to the schemeshown in FIG. 2C to generate a first NMR signal, and (2) applying asecond set of radiofrequency pulses according to the scheme shown inFIG. 2C, where phase φ₁ of the first ¹H pulse is altered by 180° togenerate a second NMR signal. Then, prior to the processing, the firstNMR signal and the second NMR signal are added and subtracted, wherebythe NMR signals are processed to generate a first NMR subspectrumderived from the subtracting which contains the peak pairs, and a secondNMR subspectrum derived from the adding which contains the additionalpeaks located centrally between the two peaks forming the peak pairs.

[0057] In an alternate embodiment, the RD 3D H,C,(C-TOCSY-CO),N,HN NMRexperiment can be modified to a RD 2D H,C,(C-TOCSY-CO,N),HN NMRexperiment, which involves applying radiofrequency pulses so that thechemical shift evolution of ¹⁵N_(i) does not occur. Then, the NMRsignals are processed to generate a two dimensional (2D) NMR spectrumwith peak pairs where (1) the chemical shift value of ¹H^(N) _(i) ismeasured in a frequency domain dimension, ω₂(¹H^(N)), and (2) thechemical shift values of ¹H^(ali) _(i−1) and ¹³C^(ali) _(i−1) aremeasured in a frequency domain dimension, ω₁(¹³C^(ali)), by thefrequency differences between the two peaks forming the peak pairs andthe frequencies at the center of the two peaks, respectively.

[0058] In an alternate embodiment, the RD 3D H,C,(C-TOCSY-CO),N,HN NMRexperiment can be modified to a RD 4D H,C,(C-TOCSY),CO,N,HN NMRexperiment which involves applying radiofrequency pulses so that thechemical shift evolution of a polypeptide backbone carbonyl carbon ofamino acid residue i−1, ¹³C′_(i−1), occurs under conditions effective togenerate NMR signals encoding the chemical shift value of ¹³C′_(l−1) ina phase sensitive manner in an indirect time domain dimension, t₄(¹³C′).Then, the NMR signals are processed to generate a four dimensional (4D)NMR spectrum with variant peak pairs where (1) the chemical shift valuesof ¹⁵N_(i), ¹H^(N) _(l) and ¹³C′_(i−1) are measured in three frequencydomain dimensions, ω₂(¹⁵N), ω₃(¹H^(N)), and ω₄(¹³C′), respectively, and(2) the chemical shift values of ¹H^(ali) _(i−1) and ¹³C^(ali) _(i−1)are measured in a frequency domain dimension, ω₁(¹³C^(ali)), by thefrequency differences between the two peaks forming the variant peakpairs and the frequencies at the center of the two peaks, respectively.

The 3D H ^(α/β),C ^(α/β),CO,HA Experiment

[0059] Another aspect of the present invention relates to a method ofconducting a reduced dimensionality (RD) three-dimensional (3D) H^(α/β),C ^(α/β),CO,HA nuclear magnetic resonance (NMR) experiment bymeasuring the chemical shift values for the following nuclei of aprotein molecule having an amino acid residue, i: (1) a β-proton ofamino acid residue i, ¹H^(β) _(l); (2) a β-carbon of amino acid residuei, ¹³C^(β) _(i); (3) an α-proton of amino acid residue i, ¹H^(α) _(l);(4) an α-carbon of amino acid residue i, ¹³C^(α) _(i); and (5) apolypeptide backbone carbonyl carbon of amino acid residue i, ¹³C′_(l).The method involves providing a protein sample and applyingradiofrequency pulses to the protein sample which effect a nuclear spinpolarization transfer where the chemical shift evolutions of ¹H^(α)_(i), ¹H^(β) _(i), ¹³C^(α) _(l), and ¹³C^(β) _(l) are connected to thechemical shift evolution of ¹³C′_(l), under conditions effective (1) togenerate NMR signals encoding the chemical shift values of ¹³C^(α) _(i),¹³C^(β) _(l) and ¹³C′_(l) in a phase sensitive manner in two indirecttime domain dimensions, t₁(¹³C^(α/β)) and t₂(¹³C′), respectively, andthe chemical shift value of ¹H^(α) _(l) in a direct time domaindimension, t₃(¹H^(α)), and (2) to cosine modulate the chemical shiftevolutions of ¹³C^(α) _(l) and ¹³C^(β) _(l) in t₁(¹³C^(α/β)) with thechemical shift evolutions of ¹H^(α) _(i) and ¹H^(β) _(l), respectively.Then, the NMR signals are processed to generate a 3D NMR spectrum withpeak pairs derived from the cosine modulating where (1) the chemicalshift values of ¹³C′_(l) and ¹H^(α) _(l) are measured in two frequencydomain dimensions, ω₂(¹³C′) and ω₃(¹H^(α)), respectively, and (2) (i)the chemical shift values of ¹H^(α) _(i) and ¹H^(β) _(i) are measured ina frequency domain dimension, ω₁(¹³C^(α/β)), by the frequencydifferences between the two peaks forming the peak pairs, and (ii) thechemical shift values of ¹³C^(α) _(i), and ¹³C^(β) _(l) are measured ina frequency domain dimension, ω₁(¹³C^(α/β)), by the frequencies at thecenter of the two peaks forming the peak pairs.

[0060] In addition, the method of conducting a RD 3D H ^(α/β), C^(α/β),CO,HA NMR experiment can involve applying radiofrequency pulsesunder conditions effective (1) to generate an additional NMR signalencoding the chemical shift values of ¹³C^(α) _(l), ¹³C^(β) _(l) and¹⁵N_(i) in a phase sensitive manner in t₁(¹³C^(α/β)) and t₂(¹⁵N) and thechemical shift value of ¹H^(α) _(i) in t₃(¹H^(α)), and (2) to avoidcosine modulating the chemical shift evolutions of ¹³C^(α) _(i) and¹³C^(β) _(i) in t₁(¹³C^(α/β)) with the chemical shift evolutions of¹H^(α) _(l) and ¹H^(β) _(i) for the additional NMR signal. Then, the NMRsignals and the additional NMR signal are processed to generate a 3D NMRspectrum with additional peaks located centrally between two peaksforming the peak pairs which measure the chemical shift values of¹³C^(α) _(i) and ¹³C^(β) _(i) along ω₁(¹³C^(α/β)). Those additionalpeaks can be derived from ¹³C^(α)and ¹³C^(β)nuclear spin polarization.One specific embodiment (3D H ^(α/β)C^(α/β)COHA) of this method isillustrated in FIG. 1E, where the applying radiofrequency pulses effectsa nuclear spin polarization transfer, where a radiofrequency pulse isused to create transverse ¹H^(α) _(i) and ¹H^(β) _(i) magnetization, and¹H^(α) _(l) and ¹H^(β) _(i) polarization is transferred to ¹³C^(α) _(i)and ¹³C^(β) _(i), to ¹³C′_(l), and back to ¹H^(α) _(l), where the NMRsignal is detected. Another specific embodiment of this method involvesapplying radiofrequency pulses by (1) applying a first set ofradiofrequency pulses according to the scheme shown in FIG. 2E togenerate a first NMR signal, and (2) applying a second set ofradiofrequency pulses according to the scheme shown in FIG. 2E, wherephase φ₁ of the first ¹H pulse is altered by 180° to generate a secondNMR signal. Then, prior to the processing, the first NMR signal and thesecond NMR signal are added and subtracted, whereby the NMR signals areprocessed to generate a first NMR subspectrum derived from thesubtracting which contains the peak pairs, and a second NMR subspectrumderived from the adding which contains the additional peaks locatedcentrally between the two peaks forming the peak pairs.

[0061] In an alternate embodiment, the RD 3D H ^(α/β),C ^(α/β),CO,HA NMRexperiment can be modified to a RD 2D H ^(α/β),C ^(α/β),(CO),HA NMRexperiment, which involves applying radiofrequency pulses so that thechemical shift evolution of ¹³C′_(i) does not occur. Then, the NMRsignals are processed to generate a two dimensional (2D) NMR spectrumwith peak pairs where (1) the chemical shift value of ¹H^(α) _(i) ismeasured in a frequency domain dimension, ω₂(¹H^(α)), and (2) (i) thechemical shift values of ¹H^(α) _(i) and ¹H^(β) _(l) are measured in afrequency domain dimension, ω₁(¹³C^(α/β)), by the frequency differencesbetween two peaks forming the peak pairs, respectively, and (ii) thechemical shift values of ¹³C^(α) _(i), and ¹³C^(β) _(i) are measured ina frequency domain dimension, ω₁(¹³C^(α/β)), by the frequencies at thecenter of the two peaks forming the peak pairs.

The 3D H ^(α/β),C ^(α/β),N,HN Experiment

[0062] A further aspect of the present invention relates to a method ofconducting a reduced dimensionality (RD) three-dimensional (3D) H^(α/β),C ^(α/β),N,HN nuclear magnetic resonance (NMR) experiment bymeasuring the chemical shift values for the following nuclei of aprotein molecule having an amino acid residue, i: (1) a β-proton ofamino acid residue i, ¹H^(β) _(l); (2) a β-carbon of amino acid residuei, ¹³C^(β) _(l); (3) an α-proton of amino acid residue i, ¹H^(β) _(l);(4) an α-carbon of amino acid residue i, ¹³C^(α) _(l); (5) a polypeptidebackbone amide nitrogen of amino acid residue i, ¹⁵N_(l); and (6) apolypeptide backbone amide proton of amino acid residue i, ¹H^(N) _(l).The method involves providing a protein sample and applyingradiofrequency pulses to the protein sample which effect a nuclear spinpolarization transfer where the chemical shift evolutions of ¹H^(α)_(i), ¹H^(β) _(i), ¹³C^(α) _(l), and ¹³C^(β) _(i) are connected to thechemical shift evolutions of ¹⁵N_(i) and ¹H^(N) _(i)under conditionseffective (1) to generate NMR signals encoding the chemical shift valuesof ¹³C^(α) _(l), ¹³C^(β) _(i) and ¹⁵N_(l) in a phase sensitive manner intwo indirect time domain dimensions, t₁(¹³C^(α/β)) and t₂(¹⁵N),respectively, and the chemical shift value of ¹H^(N) _(i) in a directtime domain dimension, t₃(¹H^(N)), and (2) to cosine modulate thechemical shift evolutions of ¹³C^(α) _(l) and ¹³C^(β) _(i) int₁(¹³C^(α/β)) with the chemical shift evolutions of ¹H^(α) _(l) and¹H^(β) _(l), respectively. Then, the NMR signals are processed togenerate a 3D NMR spectrum with peak pairs derived from the cosinemodulating where (1) the chemical shift values of ¹⁵N_(i) and ¹H^(N)_(i) are measured in two frequency domain dimensions, ω₂(¹⁵N) andω₃(¹H^(N)), respectively, and (2) (i) the chemical shift values of¹H^(α) _(l) and ¹H^(β) _(i) are measured in a frequency domaindimension, ω₁(¹³C^(α/β)), by the frequency differences between the twopeaks forming the peak pairs, and (ii) the chemical shift values of¹³C^(α) _(i), and ¹³C^(β) _(l) are measured in a frequency domaindimension, ω₁(¹³C^(α/β)), by the frequencies at the center of the twopeaks forming the peak pairs.

[0063] In addition, the method of conducting a RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment can involve applying radiofrequency pulsesunder conditions effective (1) to generate an additional NMR signalencoding the chemical shift values of ¹³C^(α) _(i), ¹³C^(β) _(i) and¹⁵N_(i) in a phase sensitive manner in t₁(¹³C^(α/β)) and t₂(¹⁵N) and thechemical shift value of ¹H^(N) _(i) in t₃(¹H^(N)), and (2) to avoidcosine modulating the chemical shift evolutions of ¹³C^(α) _(i) and¹³C^(β) _(l) in t₁(¹³C^(α/β)) with the chemical shift evolutions of¹H^(α) _(l) and ¹H^(β) _(i) for the additional NMR signal. Then, the NMRsignals and the additional NMR signal are processed to generate a 3D NMRspectrum with additional peaks located centrally between two peaksforming the peak pairs which measure the chemical shift values of¹³C^(α) _(i) and ¹³C^(β) _(l) along ω₁(¹³C^(α/β)). Those additionalpeaks can be derived from ¹³C^(α)and ¹³C^(β)nuclear spin polarization.One specific embodiment (3D H ^(α/β) C ^(α/β)NHN) of this method isillustrated in FIG. 1F, where the applying radiofrequency pulses effectsa nuclear spin polarization transfer where a radiofrequency pulse isused to create transverse ¹H^(α) _(i) and ¹H^(β) _(i) magnetization, and¹H^(α) _(i) and ¹H^(β) _(i) magnetization is transferred to ¹³C^(α) _(i)and ¹³C^(β) _(l), to ¹⁵N_(l), and to ¹H^(N) _(l), where the NMR signalis detected. Another specific embodiment of this method involvesapplying radiofrequency pulses by (1) applying a first set ofradiofrequency pulses according to the scheme shown in FIG. 2F togenerate a first NMR signal, and (2) applying a second set ofradiofrequency pulses according to the scheme shown in FIG. 2F, wherephase φ₁ of the first ¹H pulse is altered by 180° to generate a secondNMR signal. Then, prior to the processing, the first NMR signal and thesecond NMR signal are added and subtracted, whereby the NMR signals areprocessed to generate a first NMR subspectrum derived from thesubtracting which contains the peak pairs, and a second NMR subspectrumderived from the adding which contains the additional peaks locatedcentrally between the two peaks forming the peak pairs.

[0064] In an alternate embodiment, the RD 3D H ^(α/β),C ^(α/β),N,HN NMRexperiment can be modified to a RD 2D H ^(α/β),C ^(α/β),(N),HN NMRexperiment which involves applying radiofrequency pulses so that thechemical shift evolution of ¹⁵N_(i) does not occur. Then, the NMRsignals are processed to generate a two dimensional (2D) NMR spectrumwith peak pairs where (1) the chemical shift value of ¹H^(N) _(l) ismeasured in a frequency domain dimension, ω₂(¹H^(N)), and (2) (i) thechemical shift values of ¹H^(α) _(i) and ¹H^(β) _(i) are measured in afrequency domain dimension, ω₁(¹³C^(α/β)), by the frequency differencesbetween the two peaks forming the peak pairs, and (ii) the chemicalshift values of ¹³C^(α) _(i), and ¹³C^(β) _(i) are measured in afrequency domain dimension, ω₁(¹³C^(α/β)), by the frequencies at thecenter of the two peaks forming the peak pairs.

The 3D H,C,C,H-COSY Experiment

[0065] The present invention also relates to a method of conducting areduced dimensionality (RD) three-dimensional (3D) H,C,C,H-COSY nuclearmagnetic resonance (NMR) experiment by measuring the chemical shiftvalues for ¹H^(m), ¹³C^(m), ¹H^(n), and ¹³C^(n) of a protein moleculewhere m and n indicate atom numbers of two CH, CH₂ or CH₃ groups thatare linked by a single covalent carbon—carbon bond in an amino acidresidue. The method involves providing a protein sample and applyingradiofrequency pulses to the protein sample which effect a nuclear spinpolarization transfer where the chemical shift evolutions of ¹H^(m) and¹³C^(m) are connected to the chemical shift evolutions of ¹H^(n) and¹³C^(n), under conditions effective (1) to generate NMR signals encodingthe chemical shift values of ¹³C^(m) and ¹³C^(n) in a phase sensitivemanner in two indirect time domain dimensions, t₁(¹³C^(m)) andt₂(¹³C^(n)), respectively, and the chemical shift value of ¹H^(n) in adirect time domain dimension, t₃(¹H^(n)), and (2) to cosine modulate thechemical shift evolution of ¹³C^(m) in t₁(¹³C^(m)) with the chemicalshift evolution of ¹H_(m). Then, the NMR signals are processed togenerate a 3D NMR spectrum with peak pairs derived from the cosinemodulating where (1) the chemical shift values of ¹³C^(n) and ¹H^(n) aremeasured in two frequency domain dimensions, ω₂(¹³C^(n)) and ω₃(¹H^(n)),respectively, and (2) the chemical shift values of ¹H^(m) and ¹³C^(m)are measured in a frequency domain dimension, ω₁(¹³ C^(m)), by thefrequency differences between the two peaks forming the peak pairs andthe frequencies at the center of the two peaks, respectively.

[0066] In addition, the method of conducting a RD 3D H,C,C,H-COSY NMRexperiment can involve applying radiofrequency pulses under conditionseffective (1) to generate an additional NMR signal encoding the chemicalshift values of ¹³C^(m) and ¹³C^(n) in a phase sensitive manner int₁(¹³C^(m)) and t₂(¹³C^(n)) and the chemical shift value of ¹H^(n) int₃(¹H), and (2) to avoid cosine modulating the chemical shift evolutionof ¹³C^(m) in t₁(¹³C^(m)) with the chemical shift evolution of ¹H^(m)for the additional NMR signal. Then, the NMR signals and the additionalNMR signal are processed to generate a 3D NMR spectrum with additionalpeaks located centrally between two peaks forming the peak pairs whichmeasure the chemical shift value of ¹³C^(m) along ω₁(¹³C^(m)). Thoseadditional peaks can be derived from ¹³C^(m) nuclear spin polarization.One specific embodiment (3D HCCH-COSY) of this method is illustrated inFIG. 1H, where the applying radiofrequency pulses effects a nuclear spinpolarization transfer according to FIG. 1H, where a radiofrequency pulseis used to create transverse ¹H^(m) magnetization, and ¹H^(m)magnetization is transferred to ¹³C^(m), to ¹³C^(n), and to ¹H^(n),where the NMR signal is detected. Another specific embodiment of thismethod involves applying radiofrequency pulses by (1) applying a firstset of radiofrequency pulses according to the scheme shown in FIG. 2H togenerate a first NMR signal, and (2) applying a second set ofradiofrequency pulses according to the scheme shown in FIG. 2H, wherephase φ₁ of the first ¹H pulse is altered by 180° to generate a secondNMR signal. Then, prior to the processing, the first NMR signal and thesecond NMR signal are added and subtracted, whereby the NMR signals areprocessed to generate a first NMR subspectrum derived from thesubtracting which contains the peak pairs, and a second NMR subspectrumderived from the adding which contains the additional peaks locatedcentrally between the two peaks forming the peak pairs.

[0067] In an alternate embodiment, the RD 3D H,C,C,H-COSY NMR experimentcan be modified to a RD 2D H,C,(C),H-COSY NMR experiment which involvesapplying radiofrequency pulses so that the chemical shift evolution of¹³C^(n) does not occur. Then, the NMR signals are processed to generatea two dimensional (2D) NMR spectrum with peak pairs where (1) thechemical shift value of ¹H^(n) is measured in a frequency domaindimension, ω₂(¹H^(n)), and (2) the chemical shift values of ¹H^(m) and¹³C^(m) are measured in a frequency domain dimension, ω₁(¹³C^(m)), bythe frequency differences between the two peaks forming the peak pairsand the frequencies at the center of the two peaks, respectively.

The 3D H,C,C,H-TOCSY Experiment

[0068] Another aspect of the present invention relates to a method ofconducting a reduced dimensionality (RD) three-dimensional (3D)H,C,C,H-TOCSY nuclear magnetic resonance (NMR) experiment by measuringthe chemical shift values for ¹H^(m), ¹³C^(m), ¹H^(n), and ¹³C^(n) of aprotein molecule where m and n indicate atom numbers of two CH, CH₂ orCH₃ groups that may or may not be linked by a single covalentcarbon-carbon bond in an amino acid residue. The method involvesproviding a protein sample and applying radiofrequency pulses to theprotein sample which effect a nuclear spin polarization transfer wherethe chemical shift evolutions of ¹H^(m) and ¹³C^(m) are connected to thechemical shift evolutions of ¹H^(n) and ¹³C^(n), under conditionseffective (1) to generate NMR signals encoding the chemical shift valuesof ¹³C^(m) and ¹³C^(n) in a phase sensitive manner in two indirect timedomain dimensions, t₁(¹³C^(m)) and t₂(¹³C^(n)), and the chemical shiftvalue of ¹H^(n) in a direct time domain dimension, t₃(¹H^(n)), and (2)to cosine modulate the chemical shift evolution of ¹³C^(m) int₁(¹³C^(m)) with the chemical shift evolution of ¹H^(m). Then, the NMRsignals are processed to generate a 3D NMR spectrum with peak pairsderived from the cosine modulating where (1) the chemical shift valuesof ¹³C^(n) and ¹H^(n) are measured in two frequency domain dimensions,ω₂(¹³C^(n)) and ω₃(¹H^(n)), respectively, and (2) the chemical shiftvalues of ¹H^(m) and ¹³C^(m) are measured in a frequency domaindimension, ω₁(¹³C^(m)), by the frequency differences between the twopeaks forming the peak pairs and the frequencies at the center of thetwo peaks, respectively.

[0069] In addition, the method of conducting a RD 3D H,C,C,H-TOCSY NMRcan involve applying radiofrequency pulses under conditions effective(1) to generate an additional NMR signal encoding the chemical shiftvalues of ¹³C^(m) and ¹³C^(n) in a phase sensitive manner in t₁(¹³C^(m))and t₂(¹³C^(n)) and the chemical shift value of ¹H^(n) in t₃(¹H^(n)),and (2) to avoid cosine modulating the chemical shift evolution of¹³C^(m) in t₁(¹³C_(m)) with the chemical shift evolution of ¹H^(m) forthe additional NMR signal. Then, the NMR signals and the additional NMRsignal are processed to generate a 3D NMR spectrum with additional peakslocated centrally between two peaks forming the peak pairs which measurethe chemical shift value of ¹³C^(m) along ω₁(¹³C^(m)). Those additionalpeaks can be derived from ¹³C^(m) nuclear spin polarization. Onespecific embodiment (3D HCCH-TOCSY) of this method is illustrated inFIG. 1I, where the applying radiofrequency pulses effects a nuclear spinpolarization transfer where a radiofrequency pulse is used to createtransverse ¹H^(m) magnetization, and ¹H^(m) magnetization is transferredto ¹³C^(m), to ¹³C^(n), and to ¹H^(n), where the NMR signal is detected.Another specific embodiment of this method involves applyingradiofrequency pulses by (1) applying a first set of radiofrequencypulses according to the scheme shown in FIG. 2I to generate a first NMRsignal, and (2) applying a second set of radiofrequency pulses accordingto the scheme shown in FIG. 2I, where phase φ₁ of the first ¹H pulse isaltered by 180° to generate a second NMR signal. Then, prior to theprocessing, the first NMR signal and the second NMR signal are added andsubtracted, whereby the NMR signals are processed to generate a firstNMR subspectrum derived from the subtracting which contains the peakpairs, and a second NMR subspectrum derived from the adding whichcontains the additional peaks located centrally between the two peaksforming the peak pairs.

[0070] In an alternate embodiment, the RD 3D H,C,C,H-TOCSY NMRexperiment can be modified to a RD 2D H,C,(C),H-TOCSY NMR experimentwhich involves applying radiofrequency pulses so that the chemical shiftevolution of ¹³C^(n) does not occur. Then, the NMR signals are processedto generate a two dimensional (2D) NMR spectrum with peak pairs where(1) the chemical shift value of ¹H^(n) is measured in a frequency domaindimension, ω₂(¹H^(n)), and (2) the chemical shift values of ¹H^(m) and¹³C^(m) are measured in a frequency domain dimension, ω₁(¹³ C^(m)), bythe frequency differences between the two peaks forming the peak pairsand the frequencies at the center of the two peaks, respectively.

The 2D HB,CB,(CG,CD),HD Experiment

[0071] A further aspect of the present invention relates to a method ofconducting a reduced dimensionality (RD) two-dimensional (2D)HB,CB,(CG,CD),HD nuclear magnetic resonance (NMR) experiment bymeasuring the chemical shift values for the following nuclei of aprotein molecule: (1) a β-proton of an amino acid residue with anaromatic side chain, ¹H^(β); (2) β-carbon of an amino acid residue withan aromatic side chain, ¹³C^(β); and (3) a δ-proton of an amino acidresidue with an aromatic side chain, ¹H^(δ). The method involvesproviding a protein sample and applying radiofrequency pulses to theprotein sample which effect a nuclear spin polarization transfer wherethe chemical shift evolutions of ¹H^(β) and ¹³C^(β) are connected to thechemical shift evolution of ¹H^(δ), under conditions effective (1) togenerate NMR signals encoding the chemical shift value of ¹³C^(β) in aphase sensitive manner in an indirect time domain dimension,t₁(¹³C^(β)), and the chemical shift value of ¹H^(δ) in a direct timedomain dimension, t₂(¹H^(δ)), and (2) to cosine modulate the chemicalshift evolution of ¹³C^(β) in t₁(¹³C^(β)) with the chemical shiftevolution of ¹H^(β). Then, the NMR signals are processed to generate a2D NMR spectrum with a peak pair derived from the cosine modulatingwhere (1) the chemical shift value of ¹H^(δ) is measured in a frequencydomain dimension, ω₂(¹H^(δ)), and (2) the chemical shift values of¹H^(β) and ¹³C^(β) are measured in a frequency domain dimension,ω₁(¹³C^(β)), by the frequency difference between the two peaks formingthe peak pair and the frequency at the center of the two peaks,respectively.

[0072] In addition, the method of conducting a RD 2D HB,CB,(CG,CD),HDNMR experiment can involve applying radiofrequency pulses underconditions effective (1) to generate an additional NMR signal encodingthe chemical shift value of ¹³C^(β) in a phase sensitive manner int₁(¹³C^(β)) and the chemical shift value of ¹H^(δ) in t₂(¹H^(δ)), and(2) to avoid cosine modulating the chemical shift evolution of ¹³C^(β)in t₁(¹³C^(β)) with the chemical shift evolution of ¹H^(β) for theadditional NMR signal. Then, the NMR signals and the additional NMRsignal are processed to generate a 2D NMR spectrum with an additionalpeak located centrally between the two peaks forming the peak pair whichmeasure the chemical shift value of ¹³C^(β) along ω₁(¹³C). Thatadditional peak can be derived from ¹³C^(β) nuclear spin polarization.One specific embodiment (2D HBCB(CGCD)HD) of this method is illustratedin FIG. 1J, where the applying radiofrequency pulses effects a nuclearspin polarization transfer where a radiofrequency pulse is used tocreate transverse ¹H^(β) magnetization, and ¹H^(β) magnetization istransferred to ¹³C^(β), to ¹³C^(δ), and to ¹H^(δ), where the NMR signalis detected. Another specific embodiment of this method involvesapplying radiofrequency pulses by (1) applying a first set ofradiofrequency pulses according to the scheme shown in FIG. 2J togenerate a first NMR signal, and (2) applying a second set ofradiofrequency pulses according to the scheme shown in FIG. 2J, wherephase φ₁ of the first ¹H pulse is altered by 180° to generate a secondNMR signal. Then, prior to the processing, the first NMR signal and thesecond NMR signal are added and subtracted, whereby the NMR signals areprocessed to generate a first NMR subspectrum derived from thesubtracting which contains the peak pair, and a second NMR subspectrumderived from the adding which contains the additional peak locatedcentrally between the two peaks forming the peak pair.

[0073] In an alternate embodiment, the RD 2D HB,CB,(CG,CD),HD NMRexperiment can be modified to a RD 3D HB,CB,(CG),CD,HD NMR experimentwhich involves applying radiofrequency pulses so that the chemical shiftevolution of a δ-carbon of an amino acid residue with an aromatic sidechain, ¹³C^(δ) occurs under conditions effective to generate NMR signalsencoding the chemical shift value of ¹³C^(δ) in a phase sensitive mannerin an indirect time domain dimension, t₃(¹³C^(δ)). Then, the NMR signalsare processed to generate a three dimensional (3D) NMR spectrum with apeak pair where (1) the chemical shift values of ¹H^(δ) and ¹³C^(δ) aremeasured in two frequency domain dimensions, ω₂(¹H^(δ)) and ω₃(¹³C^(δ)),respectively, and (2) the chemical shift values of ¹H^(β) and ¹³C^(β)are measured in a frequency domain dimension, ω₁(¹³C^(β)), by thefrequency difference between the two peaks forming the peak pair and thefrequency at the center of the two peaks, respectively.

[0074] In an alternate embodiment, the RD 2D HB,CB,(CG,CD),HD NMRexperiment can be modified to a RD 3D HB,CB,CG,(CD),HD NMR experimentwhich involves applying radiofrequency pulses so that the chemical shiftevolution of a γ-carbon of an amino acid residue with an aromatic sidechain, ¹³C^(γ) occurs under conditions effective to generate NMR signalsencoding the chemical shift value of ¹³C^(γ) in a phase sensitive mannerin an indirect time domain dimension, t₃(¹³C^(γ)), and said processingthe NMR signals generates a three dimensional (3D) NMR spectrum with apeak pair wherein (1) the chemical shift values of ¹H^(δ) and ¹³C^(γ)are measured in two frequency domain dimensions, ω₂(¹H^(δ)) andω₃(¹³C^(γ)), respectively, and (2) the chemical shift values of ¹H^(β)and ¹³C^(β) are measured in a frequency domain dimension, ω₁(¹³C^(β)),by the frequency difference between the two peaks forming said peak pairand the frequency at the center of the two peaks, respectively.

The 2D H,C,H-COSY Experiment

[0075] The present invention also relates to a method of conducting areduced dimensionality (RD) two-dimensional (2D) H,C,H-COSY nuclearmagnetic resonance (NMR) experiment by measuring the chemical shiftvalues for ¹H^(m), ¹³C^(m), and ¹H^(n) of a protein molecule where m andn indicate atom numbers of two CH, CH₂ or CH₃ groups in an amino acidresidue. The method involves providing a protein sample and applyingradiofrequency pulses to the protein sample which effect a nuclear spinpolarization transfer where the chemical shift evolutions of ¹H^(m) and¹³C^(m) are connected to the chemical shift evolution of ¹H^(n), underconditions effective (1) to generate NMR signals encoding the chemicalshift value of ¹³C^(m) in a phase sensitive manner in an indirect timedomain dimension, t₁(¹³C^(m)), and the chemical shift value of ¹H^(n) ina direct time domain dimension, t₂(¹H^(n)), and (2) to cosine modulatethe chemical shift evolution of ¹³C^(m) in t₁(¹³C^(m)) with the chemicalshift evolution of ¹H^(m). Then, the NMR signals are processed togenerate a 2D NMR spectrum with peak pairs derived from the cosinemodulating where (1) the chemical shift value of ¹H^(n) is measured in afrequency domain dimension, ω₂(¹H^(n)), and (2) the chemical shiftvalues of ¹H^(m) and ¹³C^(m) are measured in a frequency domaindimension, ω₁(¹³C^(m)), by the frequency differences between the twopeaks forming the peak pairs and the frequencies at the center of thetwo peaks, respectively.

[0076] One specific embodiment (2D ¹H-TOCSY-HCH-COSY) of this method isillustrated in FIG. 1K, where the applying radiofrequency pulses effectsa nuclear spin polarization transfer where a radiofrequency pulse isused to create transverse ¹H^(m) magnetization, and ¹H^(m) polarizationis transferred to ¹³C^(m), to ¹H^(m), and to ¹H^(n), where the NMRsignal is detected. Although the specific embodiment illustrated in FIG.1K shows this method applied to an amino acid residue with an aromaticside chain, this method also applies to amino acid residues withaliphatic side chains. Another specific embodiment of this methodinvolves applying radiofrequency pulses according to the scheme shown inFIG. 2K.

[0077]FIG. 3 outlines which chemical shifts are correlated in thevarious NMR experiments described above.

Combinations of RD NMR Experiments

[0078] Accordingly, a suite of multidimensional RD NMR experimentsenables one to devise strategies for RD NMR-based HTP resonanceassignment of proteins.

[0079] Thus, another aspect of the present invention relates to a methodfor sequentially assigning chemical shift values of an α-proton, ¹H^(α),an α-carbon, ¹³C^(α), a polypeptide backbone amide nitrogen, ¹⁵N, and apolypeptide backbone amide proton, ¹H^(N,) of a protein molecule. Themethod involves providing a protein sample and conducting a set ofreduced dimensionality (RD) nuclear magnetic resonance (NMR) experimentson the protein sample including: (1) a RD 3D HA,CA,(CO),N,HN NMRexperiment to measure and connect chemical shift values of the α-protonof amino acid residue i−1, ¹H^(α) _(l−1), the α-carbon of amino acidresidue i−1, ¹³C^(α) _(l−1), the polypeptide backbone amide nitrogen ofamino acid residue i, ¹⁵N_(l), and the polypeptide backbone amide protonof amino acid residue i, ¹H^(N) _(i) and (2) a RD 3D HNNCAHA NMRexperiment to measure and connect the chemical shift values of theα-proton of amino acid residue i, ¹H^(α) _(l), the α-carbon of aminoacid residue i, ¹³C^(α) _(i), ¹⁵N_(l), and ¹H^(N) _(i). Then, sequentialassignments of the chemical shift values of ¹H^(α), ¹³C^(α), ¹⁵N, and¹H^(N) are obtained by (i) matching the chemical shift values of ¹H^(α)_(l−1) and ¹³C^(α) _(l−1) with the chemical shift values of ¹H^(α) _(i)and ¹³C^(α) _(i), (ii) using the chemical shift values of ¹H^(α)_(l−)and ¹³C^(α) _(i−1) to identify the type of amino acid residue i−1(Wüthrich, NMR of Proteins and Nucleic Acids, Wiley, New York (1986);Grzesiek et al., J. Biomol. NMR, 3: 185-204 (1993), which are herebyincorporated by reference in their entirety), and (iii) mapping sets ofsequentially connected chemical shift values to the amino acid sequenceof the polypeptide chain and using the chemical shift values to locatesecondary structure elements (such as α-helices and β-sheets) within thepolypeptide chain (Spera et al., J. Am. Chem. Soc., 113:5490-5492(1991); Wishart et al., Biochemistry, 31:1647-1651, which are herebyincorporated by reference in their entirety).

[0080] In one embodiment, the protein sample could, in addition to theRD 3D HA,CA,(CO),N,HN NMR experiment and the RD 3D HNNCAHA NMRexperiment, be further subjected to a RD 3D HNN<CO,CA> NMR experiment tomeasure and connect the chemical shift values of a polypeptide backbonecarbonyl carbon of amino acid residue i−1, ¹³C′_(i−1), ¹³C^(α) _(i),¹⁵N_(i), and ¹H^(N) _(l). Then, sequential assignments of the chemicalshift value of ¹³C′_(l−1), are obtained by matching the chemical shiftvalue of ¹³C^(α) _(l) measured by the RD 3D HNN<CO,CA> NMR experimentwith the sequentially assigned chemical shift values of ¹³C^(α), ¹⁵N,and ¹H^(N) measured by the RD 3D HA,CA,(CO),N,HN NMR experiment and theRD 3D HNNCAHA NMR experiment.

[0081] In another embodiment, the protein sample could, in addition tothe RD 3D HA,CA,(CO),N,HN NMR experiment and the RD 3D HNNCAHA NMRexperiment, be further subjected to (i) a RD 3D H ^(α/β),C ^(α/β),CO,HANMR experiment to measure and connect the chemical shift values of theβ-proton of amino acid residue i, ¹H^(β) _(i), the β-carbon of aminoacid residue i, ¹³C^(β) _(i), the α-proton of amino acid residue i,¹H^(α) _(l), the α-carbon of amino acid residue i, ¹³C^(α) _(i), and apolypeptide backbone carbonyl carbon of amino acid residue i, ¹³C′_(i),and (ii) a RD 3D HNN<CO,CA> NMR experiment to measure and connect thechemical shift values of ¹³C′_(i), the α-carbon of amino acid residuei+1, ¹³C^(α) _(l+1), the polypeptide backbone amide nitrogen of aminoacid residue i+1, ¹⁵N_(i+1), and the polypeptide backbone amide protonof amino acid residue i+1, ¹H^(N) _(l+1). Then, sequential assignmentsare obtained by matching the chemical shift value of ¹³C′, measured bythe RD 3D HNN<CO,CA> NMR experiment with the chemical shift value of¹³C′_(i) measured by the RD 3D H ^(α/β),C ^(α/β),CO,HA NMR experiment.

[0082] In another embodiment, the protein sample could, in addition tothe RD 3D HA,CA,(CO),N,HN NMR experiment and the RD 3D HNNCAHA NMRexperiment, be further subjected to a RD 3D H,C,(C-TOCSY-CO),N,HN NMRexperiment to measure and connect the chemical shift values of aliphaticprotons (including α-, β-, and γ-protons) of amino acid residue i−1,¹H^(ali) _(i−1), aliphatic carbons (including α-, β-, and γ-carbons) ofamino acid residue i−1, ¹³C^(ali) _(l−1), ¹⁵N_(i), and ¹H^(N) _(l).Then, sequential assignments of the chemical shift values of ¹H^(ali)_(i−1) and ¹³C^(ali) _(i−1) for amino acid residues i having uniquepairs of ¹⁵N_(l) and ₁H^(N) _(l) chemical shift values are obtained bymatching the chemical shift values of ¹H^(α) and ¹³C^(α) measured bysaid RD 3D HNNCAHA NMR experiment and RD 3D HA,CA,(CO),N,HN NMRexperiment with the chemical shift values of ¹H^(α) _(l−1) and ¹³C^(α)_(l−1) measured by said RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment andusing the ¹H^(ali) _(i−1) and ¹³C^(ali) _(i−1) chemical shift values toidentify the type of amino acid residue i−1.

[0083] In another embodiment, the protein sample could, in addition tothe RD 3D HA,CA,(CO),N,HN NMR experiment and the RD 3D HNNCAHA NMRexperiment, be further subjected to a RD 3D H,C,C,H-COSY NMR experimentor a RD 3D H,C,C,H-TOCSY NMR experiment to measure and connect thechemical shift values of ¹H^(ali) _(l) and ¹³C^(ali) _(l) of amino acidresidue i. Then, sequential assignments of the chemical shift values of¹H^(ali) _(i) and ¹³C^(ali) _(l), the chemical shift values of aγ-proton, ¹H^(γ) _(i), and a γ-carbon, ¹³C^(γ) _(i), in particular, areobtained by (i) matching the chemical shift values of ¹H^(α) _(i) and¹³C^(α) _(i) measured using the RD 3D H,C,C,H-COSY NMR experiment or theRD 3D H,C,C,H-TOCSY RD NMR experiment with the chemical shift values of¹H^(α) _(i) and ¹³C^(α) measured by the RD 3D HA, CA,(CO),N,HN NMRexperiment, the RD 3D HNNCAHA NMR experiment, and the RD 3D H ^(α/β) C^(α/β)(CO)NHN NMR experiment and (ii) using the chemical shift values of¹H^(ali) and ¹³C^(ali), the chemical shift values of ¹H^(γ) _(i) and¹³C^(γ) _(i) in particular, to identify the type of amino acid residuei.

[0084] In yet another embodiment, this method involves, in addition tothe RD 3D HA,CA,(CO),N,HN NMR experiment and the RD 3D HNNCAHA NMRexperiment, further subjecting the protein sample to a RD 3D H ^(α/β) C^(α/β)(CO)NHN NMR experiment to measure and connect the chemical shiftvalues of the β-proton of amino acid residue i−1, ¹H^(β) _(l−1), theβ-carbon of amino acid residue i−1, ¹³C^(β) _(l−1), ¹H^(α) _(i−1),¹³C^(α) _(l−1), ¹⁵N_(i), and ¹H^(N) _(i). Then, sequential assignmentsof the chemical shift values of ¹H^(β) and ¹³C^(β) are obtained by usingthe chemical shift values of ¹H^(β) _(l−1) and ¹³C^(β) _(l−1) toidentify the type of amino acid residue i−1.

[0085] In another embodiment, the protein sample could, in addition tothe RD 3D HA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHA NMRexperiment, and the RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment, befurther subjected to a RD 3D H^(α/β), C^(α/β),CO,HA NMR experiment tomeasure and connect the chemical shift values of the β-proton of aminoacid residue i, ¹H^(β) _(i), the β-carbon of amino acid residue i,¹³C^(β) _(l), ¹H^(α) _(l), ¹³C^(α) _(l), and a polypeptide backbonecarbonyl carbon of amino acid residue i, ¹³C′_(l). Then, sequentialassignments of the chemical shift value of ¹³C′_(l) are obtained bymatching the chemical shift values of ¹H^(β) _(l), ¹³C^(β) _(i), ¹H^(α)_(i), and ¹³C^(α) _(l) measured by the RD 3D H ^(α/β),C ^(α/β),CO,HA NMRexperiment with the sequentially assigned chemical shift values of¹H^(β), ¹³C^(β), ¹H^(α), ¹³C^(α), ¹⁵N, and ¹H^(N) measured by the RD 3DHA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHA NMR experiment, andthe RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment.

[0086] In another embodiment, the protein sample could, in addition tothe RD 3D HA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHA NMRexperiment, and the RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment, befurther subjected to a RD 3D H ^(α/β),C ^(α/β),N,HN NMR experiment tomeasure and connect the chemical shift values of ¹H^(β) _(i), ¹³C^(β)_(i), ¹H^(α) _(i), ¹³C^(α) _(i), ¹⁵N_(i), and ¹H^(N) _(l). Then,sequential assignments are obtained by matching the chemical shiftvalues of ¹H^(β) _(i), ¹³C^(β) _(i), ¹H^(α) _(i), and ¹³C^(α) _(i) withthe chemical shift values of ¹H^(β) _(i−1), ¹³C^(β) _(i−1), ¹H^(α)_(l−1), and ¹³C^(α) _(i−1) measured by the RD 3D H ^(α/β) C^(α/β)(CO)NHN NMR experiment.

[0087] In another embodiment, the protein sample could, in addition tothe RD 3D HA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHA NMRexperiment, and the RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment, befurther subjected to a 3D HNNCACB NMR experiment to measure and connectthe chemical shift value of ¹³C^(β) _(i), ¹³C^(α) _(i), ¹⁵N_(i), and¹H^(N) _(i). Then, sequential assignments are obtained by matching thechemical shift values of ¹³C^(β) _(i) and ¹³C^(α) _(l) measured by said3D HNNCACB NMR experiment with the chemical shift values of ¹³C^(β)_(i−1) and ¹³C^(α) _(l−1) measured by the RD 3D H ^(α/β) C ^(α/β)(CO)NHNNMR experiment.

[0088] In another embodiment, the protein sample could, in addition tothe RD 3D HA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHA NMRexperiment, and the RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment, befurther subjected to a RD 2D HB,CB,(CG,CD),HD NMR experiment to measureand connect the chemical shift values of ¹H^(β) _(i−1), ¹³C^(β) _(l−1),and a δ-proton of amino acid residue I−1 with an aromatic side chain,¹H^(δ) _(i−1). Then, sequential assignments are obtained by matching (i)the chemical shift values of ¹H^(β) _(i−1) and ¹³C^(β) _(l−1) measuredby said RD 2D HB,CB,(CG,CD),HD NMR experiment with the chemical shiftvalues of ¹H^(β) and ¹³C^(β) measured by the RD 3D H ^(α/β) C^(α/β)(CO)NHN NMR experiment, (ii) using the chemical shift values toidentify amino acid residue i as having an aromatic side chain, and(iii) mapping sets of sequentially connected chemical shift values tothe amino acid sequence of the polypeptide chain and locating amino acidresidues with aromatic side chains along the polypeptide chain.

[0089] In another embodiment, the protein sample could, in addition tothe RD 3D HA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHA NMRexperiment, and the RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment, befurther subjected to a RD 3D H,C,C,H-COSY NMR experiment or a RD 3DH,C,C,H-TOCSY NMR experiment to measure and connect the chemical shiftvalues of aliphatic protons (including α-, β-, and γ-protons) of aminoacid residue i, ¹H^(ali) _(i), and aliphatic carbons (including α-, β-,and γ-carbons) of amino acid residue i, ¹³C^(ali) _(i), of amino acidresidue i. Then, sequential assignments of the chemical shift values of¹H^(ali) _(i) and ¹³C^(ali) _(l), the chemical shift values of aγ-proton, ¹H^(γ), and a γ-carbon, ¹³C^(γ), in particular, are obtainedby (i) matching the chemical shift values of ¹H^(β) _(i), ¹³C^(β) _(i),¹H^(α) _(i), and ¹³C^(α) _(l) measured using the RD 3D H,C,C,H-COSY NMRexperiment or the RD 3D H,C,C,H-TOCSY RD NMR experiment with thechemical shift values of ¹H^(β) _(l), ¹³C^(β) _(i), ¹H^(α) _(i), and¹³C^(a) _(i) measured by the RD 3D HA,CA,(CO),N,HN NMR experiment, theRD 3D HNNCAHA NMR experiment, and the RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMRexperiment and (ii) using the chemical shift values of ¹H^(ali) and¹³C^(ali), the chemical shift values of ¹H^(γ) and ¹³C^(γ) inparticular, to identify the type of amino acid residue i.

[0090] Yet another aspect of the present invention relates to a methodfor sequentially assigning chemical shift values of a β-proton, ¹H^(β),a β-carbon, ¹³C^(β), an α-proton, ¹H^(α), an α-carbon, ¹³C^(α), apolypeptide backbone amide nitrogen, ¹⁵N, and a polypeptide backboneamide proton, ¹H^(N) _(i), of a protein molecule. The method involvesproviding a protein sample and conducting a set of reduceddimensionality (RD) nuclear magnetic resonance (NMR) experiments on theprotein sample including: (1) a RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMRexperiment to measure and connect the chemical shift values of theβ-proton of amino acid residue i−1, ¹H^(β) _(l−1), the β-carbon of aminoacid residue i−1, ¹³C^(β) _(l−1), the α-proton of amino acid residuei−1, ¹H^(α) _(i−1), the α-carbon of amino acid residue i−1, ¹³C^(α)_(l−1), the polypeptide backbone amide nitrogen of amino acid residue i,¹⁵N_(l), and the polypeptide backbone amide proton of amino acid residuei, ¹H^(N) _(i) and (2) a RD 3D H ^(α/β),C ^(α/β),N,HN NMR experiment tomeasure and connect the chemical shift values of the β-proton of aminoacid residue i, ¹H^(β) _(i), the β-carbon of amino acid residue i,¹³C^(β) _(i), the α-proton of amino acid residue i, ¹H^(α) _(i), theα-carbon of amino acid residue i, ¹³C^(α) _(i), ¹⁵N_(i), and ¹H^(N)_(i). Then, sequential assignments of the chemical shift values of¹H^(β), ¹³C^(β), ¹H^(α), ¹³C^(α), ¹⁵N, and ¹H^(N) are obtained by (i)matching the chemical shift values of the α- and β-protons of amino acidresidue i−1, ¹H^(α/β) _(i−1), and the chemical shift values of the α-and β-carbons of amino acid residue i−1, ¹³C^(α/β) _(i−1), with ¹H^(α/β)_(i) and ¹³C^(α/β) _(i), (ii) using ¹H^(α/β) _(i−1) and ¹³C^(α/β) _(i−1)to identify the type of amino acid residue i−1 (Wüthrich, NMR ofProteins and Nucleic Acids, Wiley, New York (1986); Grzesiek et al., J.Biomol. NMR, 3: 185-204 (1993), which are hereby incorporated byreference in their entirety), (iii) mapping sets of sequentiallyconnected chemical shift values to the amino acid sequence of thepolypeptide chain and using the chemical shift values to locatesecondary structure elements within the polypeptide chain (Spera et al.,J. Am. Chem. Soc., 113 :5490-5492 (1991); Wishart et al., Biochemistry,31:1647-1651, which are hereby incorporated by reference in theirentirety).

[0091] In one embodiment, the protein sample could, in addition to theRD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment and the RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment, be further subjected to a RD 3DHA,CA,(CO),N,HN NMR experiment (i) to measure and connect chemical shiftvalues of ¹H^(α) _(l−1), ¹³C^(α) _(i−1), ¹⁵N_(i), and ¹H^(N) _(i) and(ii) to distinguish between NMR signals for ¹H^(α)/¹³C^(α)and¹H^(β)/¹³C^(β)measured in the RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMRexperiment and the RD 3D H ^(α/β) C ^(α/β)N,HN NMR experiment.

[0092] In another embodiment, the protein sample could, in addition tothe RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment and the RD 3D H^(α/β),C ^(α/β),N,HN NMR experiment, be further subjected to a RD 3D H^(α/β),C ^(α/β),CO,HA NMR experiment to measure and connect the chemicalshift values of ¹H^(β) _(i), ¹³C^(β) _(i), ¹H^(α) _(l), ¹³C^(α) _(i),and a polypeptide backbone carbonyl carbon of amino acid residue i,¹³C′_(i). Then, sequential assignments of the chemical shift value of¹³C′₁ are obtained by matching the chemical shift values of ¹H^(β) _(i),¹³C^(β) _(i), ¹H^(α) _(i), and ¹³C^(α) _(i) measured by the RD 3D H^(α/β),C ^(α/β),CO,HA NMR experiment with the sequentially assignedchemical shift values of ¹H^(β), ¹³C^(β), ¹H^(α), ¹³C^(α), ¹⁵N, and¹H^(N) measured by the RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment andthe RD 3D H ^(α/β),C ^(α/β),N,HN NMR experiment.

[0093] In another embodiment, the protein sample could, in addition tothe RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment and the RD 3D H^(α/β),C ^(α/β),N,HN NMR experiment, be further subjected to a RD 3DHNN<CO,CA> NMR experiment to measure and connect the chemical shiftvalues of a polypeptide backbone carbonyl carbon of amino acid residuei−1, ¹³C′_(i−1), ¹³C^(α) _(i), ¹⁵N_(i), and ¹H^(N) _(l). Then,sequential assignments of the chemical shift value of ¹³C′_(i−1) areobtained by matching the chemical shift value of ¹³C^(α) _(i) measuredby the RD 3D HNN<CO,CA> NMR experiment with the sequentially assignedchemical shift values of ¹³C^(α), ¹⁵N, and ¹H^(N) measured by the RD 3DH ^(α/β) C ^(α/β)(CO)NHN NMR experiment and RD 3D H ^(α/β),C ^(α/β),N,HNNMR experiment.

[0094] In another embodiment, the protein sample could, in addition tothe RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment and the RD 3D H^(α/β),C ^(α/β),N,HN NMR experiment, be further subjected to (i) a RD 3DH ^(α/β),C ^(α/β),CO,HA NMR experiment to measure and connect thechemical shift values of ¹H^(β) _(i), ¹³C^(β) _(l), ¹H^(α) _(i), ¹³C^(α)_(i), and a polypeptide backbone carbonyl carbon of amino acid residuei, ¹³C′_(i) and (ii) a RD 3D HNN<CO,CA> NMR experiment to measure andconnect the chemical shift values of ¹³C′_(i), the α-carbon of aminoacid residue i+1, ¹³C^(α) _(i+1), the polypeptide backbone amidenitrogen of amino acid residue i+1, ¹⁵N_(i+1), and the polypeptidebackbone amide proton of amino acid residue i+1, ¹H^(N) _(i+1). Then,sequential assignments are obtained by matching the chemical shift valueof ¹³C′_(l) measured by said RD 3D HNN<CO,CA> NMR experiment with thechemical shift value of ¹³C′_(i) measured by the RD 3D H ^(α/β),C^(α/β),CO,HA NMR experiment.

[0095] In another embodiment, the protein sample could, in addition tothe RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment and the RD 3D H^(α/β),C ^(α/β),N,HN NMR experiment, be further subjected to a RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment to measure and connect the chemicalshift values of ¹H^(ali) _(i−1), ¹³C^(ali) _(i−1), ¹⁵N_(l), and ¹H^(N)_(l). Then, sequential assignments of the chemical shift values of¹H^(ali) _(i−1) and ¹³C^(ali) _(l−1) for amino acid residues i havingunique pairs of ¹⁵N_(i) and ¹H^(N) _(i) chemical shift values areobtained by matching the chemical shift values of ¹H^(β), ¹³C^(β),¹H^(α), and ¹³C^(α)measured by the RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMRexperiment and RD 3D H ^(α/β),C ^(α/β),N,HN NMR experiment with thechemical shift values of ¹H^(β) _(i−1), ¹³C^(β) _(i−1), ¹H^(α) _(i−1),and ¹³C^(α) _(i−1) measured by the RD 3D H,C,(C-TOCSY-CO),N,HN NMRexperiment and using the ¹H^(ali) _(l−1), and ¹³C^(ali) _(l−1) chemicalshift values to identify the type of amino acid residue i−1.

[0096] In another embodiment, the protein sample could, in addition tothe RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment and the RD 3D H^(α/β),C ^(α/β),N,HN NMR experiment, be further subjected to a 3DHNNCACB NMR experiment to measure and connect the chemical shift valueof ¹³C^(β) _(i), ¹³C^(α) _(l), ¹⁵N_(l), and ¹H^(N) _(l). Then,sequential assignments are obtained by matching the chemical shiftvalues of ¹³C^(β) _(l) and ¹³C^(α) _(i) measured by said 3D HNNCACB NMRexperiment with the chemical shift values of ¹³C^(β) _(i−1) and ¹³C^(α)_(i−1) measured by the RD 3D H+EE^(α/β)C ^(α/β)(CO)NHN NMR experiment.

[0097] In another embodiment, the protein sample could, in addition tothe RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment and the RD 3D H^(α/β),C ^(α/β),N,HN NMR experiment, be further subjected to a RD 2DHB,CB,(CG,CD),HD NMR experiment to measure and connect the chemicalshift values of ¹H^(β) _(i), ¹³C^(β) _(i), and a δ-proton of amino acidresidue i with an aromatic side chain, ¹H^(δ) _(l). Then, sequentialassignments are obtained by (i) matching the chemical shift values of¹H^(β) _(i) and ¹³C^(β) _(i) measured by said RD 2D HB,CB,(CG,CD),HD NMRexperiment with the chemical shift values of ¹H^(β) and ¹³C^(β) measuredby the RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment and the RD 3D H^(α/β),C ^(α/β),N,HN NMR experiment, (ii) using the chemical shiftvalues to identify amino acid residue i as having an aromatic sidechain, and (iii) mapping sets of sequentially connected chemical shiftvalues to the amino acid sequence of the polypeptide chain and locatingamino acid residues with aromatic side chains along the polypeptidechain (Spera et al., J. Am. Chem. Soc., 113:5490-5492 (1991); Wishart etal., Biochemistry, 31:1647-1651, which are hereby incorporated byreference in their entirety).

[0098] In another embodiment, the protein sample could, in addition tothe RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment and the RD 3D H^(α/β),C ^(α/β),N,HN NMR experiment, be further subjected to a RD 3DH,C,C,H-COSY NMR experiment or a RD 3D H,C,C,H-TOCSY NMR experiment tomeasure and connect the chemical shift values of aliphatic protons ofamino acid residue i, ¹H^(ali) _(i), and aliphatic carbons of amino acidresidue i, ¹³C^(ali) _(l), of amino acid residue i. Then, sequentialassignments of the chemical shift values of ¹H^(ali) _(i) and ¹³C^(ali)_(i), the chemical shift values of a γ-proton, ¹H^(γ) _(i), and aγ-carbon, ¹³C^(γ) _(l), in particular, are obtained by (i) matching thechemical shift values of ¹H^(β) _(i), ¹³C^(β) _(i), ¹H^(α) _(l), and¹³C^(α) _(i) measured using the RD 3D H,C,C,H-COSY NMR experiment or theRD 3D H,C,C,H-TOCSY RD NMR experiment with the chemical shift values of¹H^(β), ¹³C^(β), ¹H^(α), and ¹³C^(α) measured by the RD 3D H ^(α/β) C^(α/β)(CO)NHN NMR experiment and the RD 3D H ^(α/β),C ^(α/β),N,HN NMRexperiment, and (ii) using the chemical shift values of ¹H^(ali) _(i)and ¹³C^(ali) _(i), the chemical shift values of ¹H^(γ), and ¹³C^(γ)_(i) in particular, to identify the type of amino acid residue i.

[0099] A further aspect of the present invention involves a method forsequentially assigning the chemical shift values of aliphatic protons,¹H^(ali), aliphatic carbons, ¹³C^(ali), a polypeptide backbone amidenitrogen, ¹⁵N, and a polypeptide backbone amide proton, ¹H^(N,) of aprotein molecule. The method involves providing a protein sample andconducting a set of reduced dimensionality (RD) nuclear magneticresonance (NMR) experiments on the protein sample including: (1) a RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment to measure and connect the chemicalshift values of the aliphatic protons of amino acid residue i−1,¹H^(ali) _(l−1), the aliphatic carbons of amino acid residue i−1,¹³C^(ali) _(l−1), the polypeptide backbone amide nitrogen of amino acidresidue i, ¹⁵N_(i), and the polypeptide backbone amide proton of aminoacid residue i, ¹H^(N) _(i) and (2) a RD 3D H ^(α/β),C ^(α/β),N,HN NMRexperiment to measure and connect the chemical shift values of theβ-proton of amino acid residue i, ¹H^(β) _(l), the β-carbon of aminoacid residue i, ¹³C^(β) _(l), the α-proton of amino acid residue i,¹H^(α) _(i), the α-carbon of amino acid residue i, ¹³C^(α) _(i),¹⁵N_(i), and ¹H^(N) _(l). Then, sequential assignments of the chemicalshift values of ¹H^(ali), ¹³C^(ali), ¹⁵N, and ¹H^(N) are obtained by (i)matching the chemical shift values of the α- and β-protons of amino acidresidue i−1, ¹H^(α/β) _(l−1) and the α- and β-carbons of amino acidresidue i−1, ¹³C^(α/β) _(i−1) with the chemical shift values of ¹H^(α/β)₁ and ¹³C^(α/β) _(i) of amino acid residue i, (ii) using the chemicalshift values of ¹H^(ali) _(l−1) and ¹³C^(ali) _(i−1) to identify thetype of amino acid residue i−1 (Wüthrich, NMR of Proteins and NucleicAcids, Wiley, New York (1986); Grzesiek et al., J. Biomol. NMR, 3:185-204 (1993), which are hereby incorporated by reference in theirentirety), and (iii) mapping sets of sequentially connected chemicalshift values to the amino acid sequence of the polypeptide chain andusing the chemical shift values to locate secondary structure elementswithin the polypeptide chain (Spera et al., J. Am. Chem. Soc.,113:5490-5492 (1991); Wishart et al., Biochemistry, 31:1647-1651, whichare hereby incorporated by reference in their entirety).

[0100] In one embodiment, the protein sample could, in addition to theRD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment, be further subjected to a RD 3D H ^(α/β),C^(α/β),CO,HA NMR experiment to measure and connect the chemical shiftvalues of ¹H^(β) _(i), ¹³C^(β) _(i), ¹H^(α) _(i), ¹³C^(α) _(l), and apolypeptide backbone carbonyl carbon of amino acid residue i, ¹³C′_(i).Then, sequential assignments of the chemical shift value of ¹³C′_(i) areobtained by matching the chemical shift values of ¹H^(β) _(i), ¹³C^(β)_(i), ¹H^(α) _(i), and ¹³C^(α) _(i) measured by the RD 3D H ^(α/β),C^(α/β),CO,HA NMR experiment with the sequentially assigned chemicalshift values of ¹H^(β), ¹³C^(β), ¹H^(α), ¹³C^(α), ¹⁵N, and ¹H^(N)measured by the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3DH ^(α/β),C ^(α/β),N,HN NMR experiment.

[0101] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment, be further subjected to a RD 3D HNN<CO,CA>NMR experiment to measure and connect the chemical shift values of apolypeptide backbone carbonyl carbon of amino acid residue i−1,¹³C′_(l−1), ¹³C^(α) _(i), ¹⁵N_(l), and ¹H^(N) _(l). Then, sequentialassignments of the chemical shift value of ¹³C′_(l−1) are obtained bymatching the chemical shift value of ¹³C^(α) _(l) measured by the RD 3DHNN<CO,CA> NMR experiment with the sequentially assigned chemical shiftvalues of ¹³C^(α), ¹⁵N, and ¹H^(N) measured by the RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment.

[0102] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment, be further subjected to (i) a RD 3D H^(α/β),C ^(α/β),CO,HA NMR experiment to measure and connect the chemicalshift values of ¹H^(β) _(i), ¹³C^(β) _(i), ¹H^(α) _(i), ¹³C^(α) _(i),and a polypeptide backbone carbonyl carbon of amino acid residue i,¹³C′_(i), and (ii) a RD 3D HNN<CO,CA> NMR experiment to measure andconnect the chemical shift values of ¹³C′_(i), the α-carbon of aminoacid residue i+1, ¹³C^(α) _(l+1), the polypeptide backbone amidenitrogen of amino acid residue i+1, ¹⁵N_(i+1), and the polypeptidebackbone amide proton of amino acid residue i+1, ¹H^(N) _(l+1). Then,sequential assignments are obtained by matching the chemical shift valueof ¹³C′_(i) measured by the RD 3D HNN<CO,CA> NMR experiment with thechemical shift value of ¹³C′_(l) measured by the RD 3D H ^(α/β),C^(α/β),CO,HA NMR experiment.

[0103] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment, be further subjected to a RD 3D H ^(α/β) C^(α/β)(CO)NHN NMR experiment (i) to measure and connect the chemicalshift values of ¹H^(α/β) _(i−1), ¹³C^(α/β) _(l−1), ¹⁵N_(l), and ¹H^(N)_(l), and (ii) to identify NMR signals for ¹H^(α/β) _(i−1), ¹³C^(α/β)_(l−1), ¹⁵N_(l), and ¹H^(N) _(i) in the RD 3D H,C,(C-TOCSY-CO),N,HN NMRexperiment.

[0104] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment, be further subjected to a RD 3DHA,CA,(CO),N,HN NMR experiment (i) to measure and connect chemical shiftvalues of ¹H^(α) _(i−1), ¹³C^(α) _(l−1), ¹⁵N_(i), and ¹H^(N) _(i) and(ii) to identify NMR signals for ¹H^(α) and ¹³C^(α) in the RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment.

[0105] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment, be further subjected to a 3D HNNCACB NMRexperiment to measure and connect the chemical shift value of ¹³C^(β)_(i), ¹³C^(α) _(l), ¹⁵N_(l), and ¹H^(N) _(i). Then, sequentialassignments are obtained by matching the chemical shift values of¹³C^(β) _(i) and ¹³C^(a) _(i) measured by said 3D HNNCACB NMR experimentwith the chemical shift values of ¹³C^(β) _(i−1) and ¹³C^(α) _(l−1)measured by the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment.

[0106] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment, be further subjected to a RD 2DHB,CB,(CG,CD),HD NMR experiment to measure and connect the chemicalshift values of ¹H^(β) _(i), ¹³C^(β) _(i), and a δ-proton of amino acidresidue i with an aromatic side chain, ¹H^(δ) _(i). Then, sequentialassignments are obtained by matching the chemical shift values of ¹H^(β)_(i) and ¹³C^(β) _(i) measured by said RD 2D HB,CB,(CG,CD)ND NMRexperiment with the chemical shift values of ¹H^(β) and ¹³C^(β) measuredby the RD 3D H ^(α/β),C ^(α/β),N,HN NMR experiment and the RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment, using the chemical shift values toidentify amino acid residue i as having an aromatic side chain, andmapping sets of sequentially connected chemical shift values to theamino acid sequence of the polypeptide chain and locating amino acidresidues with aromatic side chains along the polypeptide chain.

[0107] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment, be further subjected to a RD 3D H,C,C,H—COSYNMR experiment or a RD 3D H,C,C,H-TOCSY NMR experiment to measure andconnect the chemical shift values of aliphatic protons of amino acidresidue i, ¹H^(ali) _(l), and aliphatic carbons of amino acid residue i,¹³C^(ali) _(l). Then, sequential assignments of the chemical shiftvalues of ¹H^(ali) _(i) and ¹³C^(ali) _(l), the chemical shift values ofa γ-proton, ¹H^(γ) _(i), and a γ-carbon, ¹³C^(γ) _(l), in particular,are obtained by (i) matching the chemical shift values of ¹H^(ali) _(l)and ¹³C^(ali) _(l) measured using the RD 3D H,C,C,H-COSY NMR experimentor the RD 3D H,C,C,H-TOCSY NMR experiment with the chemical shift valuesof ¹H^(ali) and ¹³C^(ali) measured by the RD 3D H,C,(C-TOCSY-CO),N,HNNMR experiment and RD 3D H ^(α/β),C ^(α/β),N,HN NMR experiment, and (ii)using the chemical shift values of ¹H^(ali) _(i) and ¹³C^(ali) _(i), thechemical shift values of ¹H^(γ) _(i) and ¹³C^(γ) _(i) in particular, toidentify the type of amino acid residue i.

[0108] The present invention also relates to a method for sequentiallyassigning chemical shift values of aliphatic protons, ¹H^(ali),aliphatic carbons, ¹³C^(ali), a polypeptide backbone amide nitrogen,¹⁵N, and a polypeptide backbone amide proton, ¹H^(N), of a proteinmolecule. The method involves providing a protein sample and conductinga set of reduced dimensionality (RD) nuclear magnetic resonance (NMR)experiments on the protein sample including: (1) a RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment to measure and connect the chemicalshift values of the aliphatic protons of amino acid residue i−1,¹H^(ali) _(i−1), the aliphatic carbons of amino acid residue i−1,¹³C^(ali) _(i−1), the polypeptide backbone amide nitrogen of amino acidresidue i, ¹⁵N_(i), and the polypeptide backbone amide proton of aminoacid residue i, ¹H^(N) _(l) and (2) a RD 3D HNNCAHA NMR experiment tomeasure and connect the chemical shift values of the α-proton of aminoacid residue i, ¹H^(α) _(i), the α-carbon of amino acid residue i,¹³C^(α) _(i), ¹⁵N_(i), and ¹H^(N) _(i). Then, sequential assignments ofthe chemical shift values of ¹H^(ali), ¹³C^(ali), ¹⁵N, and ¹H^(N) areobtained by (i) matching the chemical shift values of the α-proton ofamino acid residue i−1, ¹H^(α) _(i−1) and the α-carbon of amino acidresidue i−1, ¹³C^(α) _(i−1) with the chemical shift values of ¹H^(α)_(l) and ¹³C^(α) _(l), (ii) using the chemical shift values of ¹H^(ali)_(l−1) and ¹³C^(ali) _(l−1) to identify the type of amino acid residuei−1 (Wüthrich, NMR of Proteins and Nucleic Acids, Wiley, New York(1986); Grzesiek et al., J. Biomol. NMR, 3: 185-204 (1993), which arehereby incorporated by reference in their entirety), and (iii) mappingsets of sequentially connected chemical shift values to the amino acidsequence of the polypeptide chain and using the chemical shift values tolocate secondary structure elements within the polypeptide chain (Speraet al., J. Am. Chem. Soc., 113:5490-5492 (1991); Wishart et al.,Biochemistry, 31:1647-1651, which are hereby incorporated by referencein their entirety).

[0109] In one embodiment, the protein sample could, in addition to theRD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D HNNCAHA NMRexperiment, be further subjected to a RD 3D H ^(α/β),C^(α/β),CO,HA NMRexperiment to measure and connect the chemical shift values of aβ-proton of amino acid residue i, ¹H^(β) _(i), a β-carbon of amino acidresidue i, ¹³C^(β) _(i), ¹H^(α) _(l), ¹³C^(α) _(l), and a polypeptidebackbone carbonyl carbon of amino acid residue i, ¹³C′^(i). Then,sequential assignments of the chemical shift value of ¹³C′_(i) areobtained by matching the chemical shift values of ¹H^(β) _(i), ¹³C^(β)_(i), ¹H^(α) _(i), and ¹³C^(α) _(l) measured by the RD 3D H ^(α/β),C^(α/β),CO,HA NMR experiment with the sequentially assigned chemicalshift values of ¹H^(β), ¹³C^(β), ¹H^(α), ¹³C^(α), ¹⁵N, and ¹H^(N)measured by the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3DHNNCAHA NMR experiment.

[0110] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D HNNCAHA NMRexperiment, be further subjected to a RD 3D HNN<CO,CA> NMR experiment tomeasure and connect the chemical shift values of a polypeptide backbonecarbonyl carbon of amino acid residue i−1, ¹³C′_(i−1), ¹³C^(α) _(i),¹⁵N_(l), and ¹H^(N) _(l). Then, sequential assignments of the chemicalshift value of ¹³C′_(i−1) are obtained by matching the chemical shiftvalue of ¹³C^(α) _(l) measured by the RD 3D HNN<CO,CA> NMR experimentwith the sequentially assigned chemical shift values of ¹³C^(α), ¹⁵N,and ¹H^(N) measured by the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experimentand the RD 3D HNNCAHA NMR experiment.

[0111] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D HNNCAHA NMRexperiment, be further subjected to (i) a RD 3D H ^(α/β),C ^(α/β),CO,HANMR experiment to measure and connect the chemical shift values of aβ-proton of amino acid residue i, ¹H^(β) _(i), a β-carbon of amino acidresidue i, ¹³C^(β) _(l), the α-proton of amino acid residue i, ¹H^(α)_(i), the α-carbon of amino acid residue i, ¹³C^(α) _(l), and apolypeptide backbone carbonyl carbon of amino acid residue i, ¹³C′_(i),and (ii) a RD 3D HNN<CO,CA> NMR experiment to measure and connect thechemical shift values of ¹³C′_(l), an α-carbon of amino acid residuei+1, ¹³C^(α) _(i+1), a polypeptide backbone amide nitrogen of amino acidresidue i+1, ¹⁵N_(l+1), and the polypeptide backbone amide proton ofamino acid residue i+1, ¹H^(N) _(i+1). Then, sequential assignments areobtained by matching the chemical shift value of ¹³C′_(i) measured bythe RD 3D HNN<CO,CA> NMR experiment with the chemical shift value of¹³C′_(i) measured by the RD 3D H ^(α/β),C ^(α/β),CO,HA NMR experiment.

[0112] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D HNNCAHA NMRexperiment, be further subjected to a RD 3D H^(α/β)C^(α/β)(CO)NHN NMRexperiment (i) to measure and connect the chemical shift values of theα- and β-protons of amino acid residue i−1, ¹H^(α/β) _(i−1), the α- andβ-carbons of amino acid residue i−1, ¹³C^(α/β) _(i−1), ¹⁵N_(l), and¹H^(N) _(i), and (ii) to distinguish NMR signals for the chemical shiftvalues of ¹H^(β) _(i−1), ¹³C^(β) _(l−1), ¹H^(α) _(i−1), and ¹³C^(α)_(l−1) measured by the RD 3D H ^(α/βC) ^(α/β)(CO)NHN NMR experiment fromNMR signals for the chemical shift values of ¹H^(ali) _(i−1) and¹³C^(ali) _(i−1) other than ¹H^(α/β) _(l−1) and ¹³C^(α/β) _(i−1)measured by the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment.

[0113] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D HNNCAHA NMRexperiment, be further subjected to a RD 3D H ^(α/β),C ^(α/β),N,HN NMRexperiment to measure and connect the chemical shift values of ¹H^(β)_(l), ¹³C^(β) _(l), ¹H^(α) _(i), ¹³C^(α) _(l), ¹⁵N^(l), and ¹H^(N) _(i).Then, sequential assignments are obtained by matching the chemical shiftvalues of ¹H^(β) _(i), ¹³C^(β) _(i), ¹H^(α) _(l), and ¹³C^(α) _(i)measured by said RD 3D H ^(α/β),C ^(α/β),N,HN NMR experiment with thechemical shift values of ¹H^(β) _(i−1), ¹³C^(β) _(i−1), ¹H^(α) _(i−1),and ¹³C^(α) _(l−1) measured by the RD 3D H,C(C-TOCSY-CO),N,HN NMRexperiment.

[0114] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D HNNCAHA NMRexperiment, be further subjected to a 3D HNNCACB NMR experiment tomeasure and connect the chemical shift values of ¹³C^(β) _(l), ¹³C^(α)_(i), ¹⁵N_(l), and ¹H^(N) _(i). Then, sequential assignments areobtained by matching the chemical shift values of ¹³C^(β) _(l) and¹³C^(α) _(i) measured by said 3D HNNCACB NMR experiment with thechemical shift values of ¹³C^(β) _(i−1) and ¹³C^(α) _(i−1) measured bythe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment.

[0115] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D HNNCAHA NMRexperiment, be further subjected to a RD 2D HB,CB,(CG,CD),HD NMRexperiment to measure and connect the chemical shift values of ¹H^(β)_(i), ¹³C^(β) _(i), and a δ-proton of amino acid residue i with anaromatic side chain, ¹H^(δ) _(i). Then, sequential assignments areobtained by matching the chemical shift values of ¹H^(β) _(l) and¹³C^(β) _(i) measured by said RD 2D HB,CB,(CG,CD),HD NMR experiment withthe chemical shift values of ¹H^(β) and ¹³C^(β) measured by the RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment, using the chemical shift values toidentify amino acid residue i as having an aromatic side chain, andmapping sets of sequentially connected chemical shift values to theamino acid sequence of the polypeptide chain and by locating amino acidresidues with aromatic side chains along the polypeptide chain.

[0116] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D HNNCAHA NMRexperiment, be further subjected to a RD 3D H,C,C,H-COSY NMR experimentor a RD 3D H,C,C,H-TOCSY NMR experiment to measure and connect thechemical shift values of aliphatic protons of amino acid residue i,¹H^(ali) _(i), and aliphatic carbons of amino acid residue i, ¹³C^(ali)_(l). Then, sequential assignments of the chemical shift values of¹H^(ali) _(l) and ¹³C^(ali) _(l), the chemical shift values of aγ-proton, ¹H^(γ) _(i), and a γ-carbon, ¹³C^(γ) _(l), in particular, areobtained by (i) matching the chemical shift values of ¹H^(ali) and¹³C^(ali) measured using the RD 3D H,C,C,H-COSY NMR experiment or the RD3D H,C,C,H-TOCSY NMR experiment with the chemical shift values of ¹H^(β)_(l), ¹³C^(β) _(l), ¹H^(α) _(i), and ¹³C^(α) _(l) measured by the RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D HNNCAHA NMRexperiment, and (ii) using the chemical shift values of ¹H^(ali) _(i)and ¹³C^(ali) _(i), the chemical shift values of ¹H^(γ) _(l) and ¹³C⁶⁵_(i) in particular, to identify the type of amino acid residue i.

[0117] Another aspect of the present invention involves a method forobtaining nearly complete assignments of chemical shift values of ¹H,¹³C and ¹⁵N of a protein molecule (excluding only chemical shift valuesof ¹³C^(δ) and ¹⁵N^(ε2) of glutamines, of ¹³C^(γ) and ¹⁵N^(δ2) ofasparagines, of ¹³C^(ε3), ¹H^(ε3), ¹³C^(ζ2), ¹H^(ζ2), ¹³C^(ζ3), ¹H^(ζ3),¹³C^(η2), and ¹H^(η2) groups of tryptophans, of ¹³C^(ε) and ¹H^(ε) ofmethionines, and of labile sidechain protons that exchange rapidly withthe protons of the solvent water) (Yamazaki et al., J. Am. Chem. Soc.,115:11054-11055 (1993), which is hereby incorporated by reference in itsentirety), which are required for the determination of the tertiarystructure of a protein in solution (Wüthrich, NMR of Proteins andNucleic Acids, Wiley, New York (I 986), which is hereby incorporated byreference in its entirety). The method involves providing a proteinsample and conducting four reduced dimensionality (RD) nuclear magneticresonance (NMR) experiments on the protein sample, where (1) a firstexperiment is selected from the group consisting of a RDthree-dimensional (3D) H ^(α/β) C ^(α/β)(CO)NHN NMR experiment, a RD 3DHA,CA,(CO),N,HN NMR experiment, and a RD 3D H,C,(C-TOCSY-CO),N,HN NMRexperiment for obtaining sequential correlations of chemical shiftvalues; (2) a second experiment is selected from the group consisting ofa RD 3D HNNCAHA NMR experiment, a RD 3D H ^(α/β),C ^(α/β),N,HN NMRexperiment, and a RD 3D HNN<CO,CA> NMR experiment for obtainingintraresidue correlations of chemical shift values; (3) a thirdexperiment is a RD 3D H,C,C,H-COSY NMR experiment for obtainingassignments of aliphatic and aromatic sidechain chemical shift values;and (4) a fourth experiment is a RD 2D HB,CB,(CG,CD),HD NMR experimentfor obtaining assignments of aromatic sidechain chemical shift values.

[0118] In one embodiment of this method, the protein sample could befurther subjected to a RD 2D H,C,H-COSY NMR experiment for obtainingassignments of aliphatic and aromatic sidechain chemical shift values.

[0119] In another embodiment of this method, the first experiment is theRD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment and the second experimentis the RD 3D HNNCAHA NMR experiment.

[0120] In another embodiment, the protein sample could, in addition tothe RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment, RD 3D HNNCAHA NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHA,CA,(CO),N,HN NMR experiment to distinguish between NMR signals for¹H^(α)/¹³C^(α) and ¹H^(β)/³C^(β) from the RD 3D H ^(α/β) C ^(α/β)(CO)NHNNMR experiment.

[0121] In another embodiment, the protein sample could, in addition tothe RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment, RD 3D HNNCAHA NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment to obtain assignments of chemicalshift values of ¹H^(ali) and ¹³C^(ali).

[0122] In another embodiment, the protein sample could, in addition tothe RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment, RD 3D HNNCAHA NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D H^(α/β),C ^(α/β),N,HN NMR experiment to obtain assignments of chemicalshift values of ¹H^(β) and ¹³C^(β).

[0123] In another embodiment, the protein sample could, in addition tothe RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment, RD 3D HNNCAHA NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHNN<CO,CA> NMR experiment to obtain assignments of chemical shift valuesof polypeptide backbone carbonyl carbons, ¹³C′.

[0124] In another embodiment, the protein sample could, in addition tothe RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment, RD 3D HNNCAHA NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D H^(α/β),C ^(α/β),CO,HA NMR experiment to obtain assignments of chemicalshift values of polypeptide backbone carbonyl carbons, ¹³C′.

[0125] In another embodiment, the protein sample could, in addition tothe RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment, RD 3D HNNCAHA NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHNN<CO,CA> NMR experiment and a RD 3D H ^(α/β),C ^(α/β),CO,HA NMRexperiment to obtain assignments of chemical shift values of ¹³C′.

[0126] In another embodiment, the protein sample could, in addition tothe RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment, RD 3D HNNCAHA NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,C,H-TOCSY NMR experiment to obtain assignments of chemical shiftvalues of ¹H and ¹³C of aliphatic sidechains.

[0127] In another embodiment, the protein sample could, in addition tothe RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment, RD 3D HNNCAHA NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,C,H-TOCSY NMR experiment to obtain assignments of chemical shiftvalues of ¹H and ¹³C of aromatic sidechains.

[0128] In another embodiment, the protein sample could, in addition tothe RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment, RD 3D HNNCAHA NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a 3D HNNCACBNMR experiment to obtain assignments of chemical shift values of¹³C^(β).

[0129] In yet another embodiment of this method, the first experiment isthe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the second experimentis the RD 3D HNNCAHA NMR experiment.

[0130] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNNCAHA NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHA,CA,(CO),N,HN NMR experiment to identify NMR signals for¹H^(α)/¹³C^(α) in the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment.

[0131] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNNCAHA NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D H^(α/β),C ^(α/β),N,HN NMR experiment to obtain assignments of chemicalshift values of ¹H^(β) and ¹³C^(β).

[0132] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNNCAHA NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHNN<CO,CA> NMR experiment to obtain assignments of chemical shift valuesof polypeptide backbone carbonyl carbons, ¹³C′.

[0133] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNNCAHA NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D H^(α/β),C ^(α/β),CO,HA NMR experiment to obtain assignments of chemicalshift values of polypeptide backbone carbonyl carbons, ¹³C′.

[0134] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNNCAHA NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHNN<CO,CA> NMR experiment and a RD 3D H ^(α/α),C ^(α/β),CO,HA NMRexperiment to obtain assignments of chemical shift values of ¹³C′.

[0135] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNNCAHA NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,C,H-TOCSY NMR experiment to obtain assignments of chemical shiftvalues of ¹H and ¹³C of aliphatic sidechains.

[0136] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNNCAHA NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,C,H-TOCSY NMR experiment to obtain assignments of chemical shiftvalues of ¹H and ¹³C of aromatic sidechains.

[0137] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNNCAHA NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a 3D HNNCACBNMR experiment to obtain assignments of chemical shift values of¹³C^(β).

[0138] In yet another embodiment of this method, the first experiment isthe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the second experimentis the RD 3D H ^(α/β),C ^(α/β),N,HN NMR experiment.

[0139] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHA,CA,(CO),N,HN NMR experiment to identify NMR signals for ¹H^(α) and¹³C^(α) in the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment.

[0140] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D H^(α/β) C ^(α/β)(CO)NHN NMR experiment to identify NMR signals for¹H^(α/β) and ¹³C^(α/β) in the RD 3D H,C,(C-TOCSY-CO),N,HN NMRexperiment.

[0141] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHNN<CO,CA> NMR experiment to obtain assignments of chemical shift valuesof polypeptide backbone carbonyl carbons, ¹³C′.

[0142] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D H ^(α/β),C ^(α/β),N,HN NMR experiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D H^(α/β),C ^(α/β),CO,HA NMR experiment to obtain assignments of chemicalshift values of polypeptide backbone carbonyl carbons, ¹³C′.

[0143] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHNN<CO,CA> NMR experiment and a RD 3D H ^(α/β),C ^(α/β),CO,HA NMRexperiment to obtain assignments of chemical shift values of ¹³C′.

[0144] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,C,H-TOCSY NMR experiment to obtain assignments of chemical shiftvalues of ¹H and ¹³C of aliphatic sidechains.

[0145] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,C,H-TOCSY NMR experiment to obtain assignments of chemical shiftvalues of ¹H and ¹³C of aromatic sidechains.

[0146] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D H ^(α/β),C^(α/β),N,HN NMR experiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a 3D HNNCACBNMR experiment to obtain assignments of chemical shift values of¹³C^(β).

[0147] In yet another embodiment of this method, the first experiment isthe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the second experimentis the RD 3D HNN<CO,CA> NMR experiment.

[0148] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNN<CO,CA> NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHA,CA,(CO),N,HN NMR experiment to identify NMR signals for ¹H^(α) and¹³C^(α) in the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment.

[0149] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNN<CO,CA> NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D H^(α/β) C ^(α/β)(CO)NHN NMR experiment to identify NMR signals for¹H^(α/β) and ¹³C^(α/β) in the RD 3D H,C,(C-TOCSY-CO),N,HN NMRexperiment.

[0150] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNN<CO,CA> NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D H^(α/β),C ^(α/β),CO,HA NMR experiment to obtain assignments of chemicalshift values of polypeptide backbone carbonyl carbons, ¹³C′.

[0151] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNN<CO,CA> NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,C,H-TOCSY NMR experiment to obtain assignments of chemical shiftvalues of ¹H and ¹³C of aliphatic sidechains.

[0152] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNN<CO,CA> NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,C,H-TOCSY NMR experiment to obtain assignments of chemical shiftvalues of ¹H and ¹³C of aromatic sidechains.

[0153] In another embodiment, the protein sample could, in addition tothe RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNN<CO,CA> NMRexperiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a 3D HNNCACBNMR experiment to obtain assignments of chemical shift values of¹³C^(β).

[0154] In addition, the above-described method for obtaining assignmentsof chemical shift values of ¹H, ¹³C and ¹⁵N of a protein molecule caninvolve further subjecting the protein sample to nuclear Overhausereffect spectroscopy (NOESY) (Wüthrich, NMR of Proteins and NucleicAcids, Wiley, New York (1986), which is hereby incorporated by referencein its entirety), to NMR experiments that measure scalar couplingconstants (Eberstadt et al., Angew. Chem. Int. Ed. Engl., 34:1671-1695(1995); Cordier et al., J. Am. Chem. Soc., 121:1601 -1602 (1999), whichare hereby incorporated by reference in their entirety), or to NMRexperiments that measure residual dipolar coupling constants(Prestegard, Nature Struct. Biol., 5:517-522 (1998); Tjandra et al.,Science, 278:1111-1114 (1997), which are hereby incorporated byreference in their entirety), to deduce the tertiary fold or tertiarystructure of the protein molecule.

[0155] A standard set of nine experiments (labeled with asterisks inTable 2) can be employed for obtaining nearly complete resonanceassignments of proteins including aliphatic and aromatic side chain spinsystems. TABLE 2 NMR experiments acquired^(a) for the 8.5 kDa protein“Z-domain” Minimal^(c) measurement time [h] Indirect^(b) t_(max);Complex Measurement with/without Experiment dimension(s) points [ms]time [h] central peak 3D spectra for sequential backbone connectivities:**H ^(α/β) C ^(α/β)(CO)NHN ω₁(¹³C^(α/β)) 6.3; 95 9.2 4.6/2.3 ω₂(¹⁵N)21.5; 28 HACA(CO)NHN ω₁(¹³C^(α)) 6.5; 54 54/27^(d) 5.4/2.7 ω₂(¹⁵N) 21.5;28 HC(C-TOCSY-CO)NHN^(d) ω₁(¹³C^(α/β)) 6.1; 90 17.9 4.5/2.3 ω₂(¹⁵N)21.5; 28 3D spectra for intraresidual backbone connectivities: **HNNCAHAω₁(¹³C^(α)) 6.6;51 5.0 2.5/n.a. ω₂(¹⁵N) 21.5; 28 *H ^(α/β) C ^(α/β)COHAω₁(¹³C^(α/β)) 6.3; 95 10.0 5.0/2.5 ω₂(¹³C = O) 17.8; 32 H ^(α/β) C^(α/β)NHN ω₁(¹³C^(α/β)) 6.0; 90 17.1 4.3/2.2 ω₂(¹⁵N) 21.5; 28 *HNNCACBω₁(¹³C^(α/β)) 6.6; 56 8.0 n.a./2.0 ω₂(¹⁵N) 21.5; 28 3D spectrum forintra- and sequential backbone connectivities: *HNN<CO,CA> ω₁(¹³C = O)8.0/16.0^(e); 54 5.5 2.8/n.a. ω₂(¹⁵N) 21.5; 28 3D spectra for assignmentof aliphatic resonances: **HCCH-COSY ω₁(¹³C) 6.3; 95 6.2 3.1/1.6 ω₂(¹³C)6.4; 20 **HCCH-TOCSY^(f) ω₁(¹³C) 6.3; 95 7.0 3.5/1.7 ω₂(¹³C) 6.4; 20 2Dspectra for assignment of aromatic resonances: **HBCB(CGCD)HD ω₁(¹³C)6.3; 95 5.3  0.1/0.05 **¹H-TOCSY-HCH-COSY^(f) ω₁(¹³C) 15; 150 3.40.2/n.a. #with an asterisk (* or **), and those spectra which can bedesignated a “minimal” set are labeled with a double-asterisk (**). #itsentirety) from ¹³C magnetization requires recording of two data setsthat are added and subtracted to generate subspectra I and II.

[0156] For larger proteins, complementary recording of highly sensitive3D HACA(CO)NHN promises (i) to yield spin systems which escape detectionin H ^(α/β) C ^(α/β)(CO)NHN, and (ii) to offer the distinction of α- andβ-moiety resonances by comparison with H ^(α/β) C ^(α/β)(CO)NHN.Furthermore, employment of 50% random fractional protein deuteration(LeMaster, Annu. Rev. Biophys. Biophys. Chem., 19:43-266 (1990);Nietlispach et al., J. Am. Chem. Soc., 118:407-415 (1996); Shan et al.,J. Am. Chem. Soc., 118:6570-6579 (1996); Leiting et al., Anal. Biochem.,265:351-355 (1998); Hochuli et al., J. Biomol. NMR, 17:33-42 (2000),which are hereby incorporated by reference in their entirety) incombination with the standard suite of NMR experiments (or transverserelaxation-optimized spectroscopy (TROSY) versions thereof) isattractive. The impact of deuteration for recording 4DH^(α/β)C^(α/β)(CO)NHN for proteins reorienting with correlation times upto around 20 ns (corresponding to a molecular weight around 30 kDa atambient T) has been demonstrated (Nietlispach et al., J. Am. Chem. Soc.,1 18:407-415 (1996), which is hereby incorporated by reference in itsentirety). Accordingly, 3D H ^(α/β) C ^(α/β)(CO)NHN can be expected tomaintain its pivotal role for obtaining complete resonance assignments(FIG. 4) for deuterated proteins at least up to about that size.Furthermore, protein deuteration offers the advantage that HNNCACB,which can be expected to become significantly less sensitive thanHNNCAHA for larger non-deuterated systems, (Szyperski et al., J. Biomol.NMR, 11:387-405 (1998), which is hereby incorporated by reference in itsentirety) can be kept to recruit ¹³C^(β) chemical shifts for sequentialassignment (Shan et al., J. Am. Chem. Soc., 118:6570-6579 (1996), whichis hereby incorporated by reference in its entirety).

[0157] If solely chemical shifts are considered, the unambiguousidentification of peaks pairs is more involved whenever multiple peakpairs with degenerate chemical shifts in the other dimensions arepresent. The acquisition of the corresponding central peaks addressesthis complication in a conceptually straightforward fashion. However, itis important to note that pairs of peaks generated by a chemical shiftin-phase splitting have quite similar intensity. In contrast, peak pairsarising from different moieties, possible located in different aminoacid residues, most often do not show similar intensity. This is becausethe nuclear spin relaxation times, which determine the peak intensities,vary within each residue as well as along the polypeptide chain. One maythus speak of a “nuclear spin relaxation time labeling” of peak pairs,which makes their identification an obvious task in most cases.

[0158] Using cryogenic probes can reduce NMR measurement times by abouta factor of 10 or more (Flynn et al., J. Am Chem. Soc., 122:4823-4824(2000), which is hereby incorporated by reference in its entirety).Hence, the standard set of nine experiments (Table 2) could have beenrecorded with the same signal-to-noise ratios measured for the presentstudy in about 6 hours using a cryogenic probe, i.e., the highsensitivity of cryogenic probes shifts even the recording of RD NMRexperiments entirely into the sampling limited data acquisition regime.In view of this dramatic reduction in spectrometer time demand,minimally achievable RD NMR measurement times are of keen interest(Table 2) to be able to adapt the NMR measurement times to sensitivityrequirements in future HTP endeavours.

[0159] If the standard set of experiments would have been recorded witha single transient per increment, 21.8 hours of spectrometer measurementtime would have been required (Table 2). This is still about 3.5 timeslonger than the 6 hours alluded to above, which would be needed on acurrently available cryogenic probe. To further reduce the measurementtime, and in view of the aforementioned ‘spin relaxation timelabeling’of peak pairs', one may then decide to also discard the use of¹³C-steady state magnetization for central peak detection. This wouldlead to a diminished requirement of 15.5 hours for the standard, or 8.1hours for the minimal set of experiments (four projected 4D and twoprojected 3D spectra; Table 2). Hence, the measurement time of theminimal set of RD NMR experiments (which provides complete resonanceassignments for Z-domain) could actually be neatly adjusted to thesensitivity requirements of a cryogenic probe.

[0160] Although RD NMR was proposed in 1993 (Szyperski et al., J.Biomol. NMR, 3:127-132 (1993); Szyperski et al., J. Am. Chem. Soc.,115:9307-9308 (1993), which are hereby incorporated by reference intheir entirety), its wide-spread use has been delayed by the moredemanding spectral analysis when compared to conventional TR NMR. Inparticular, the necessity to extract chemical shifts from in-phasesplittings suggests that strong computer support is key for employmentof RD NMR on a routine basis. This can be readily addressed by usingautomated resonance assignment software for automated analysis of RD TRNMR data.

[0161] In conclusion, the joint employment of RD NMR spectroscopy,cryogenic probes, and automated backbone resonance assignment will allowone to determine a protein's backbone resonance assignments andsecondary structure in a short time.

EXAMPLES

[0162] The following examples are provided to illustrate embodiments ofthe present invention but are by no means intended to limit its scope.

Example 1 Sample Preparation

[0163] NMR measurements were performed using a 1 mM solution ofuniformly ¹³C/¹⁵N enriched “Z-domain” of the Staphylococcal protein A(Tashiro et al., J. Mol. Biol., 272:573-590 (1997); Lyons et al.,Biochemistry, 32:7839-7845 (1993), which are hereby incorporated byreference in their entirety) dissolved in 90% D₂O/10% H₂O (20 mM K—PO₄)at pH=6.5.

Example 2 NMR Spectroscopy

[0164] Multidimensional NMR experiments (FIG. 1; Table 1) were recordedfor a 1 mM solution of the 8.5 kDa protein “Z-domain” at a temperatureof 25° C. The spectra (Table 2) were assigned, and the chemical shiftsobtained from RD NMR (Table 3) were in very good agreement with thosepreviously determined at 30° C. using conventional triple resonance (TR)NMR spectroscopy (Tashiro et al., J. Mol. Biol., 272:573-590 (1997);Lyons et al., Biochemistry, 32:7839-7845 (1993), which are herebyincorporated by reference in their entirety). TABLE 3 Chemical shifts ofthe Z-domain (in ppm relative to DSS) determined at T = 25° C. ResidueCO N HN Hα(Cα) Hβ(Cβ) others Q (−5) 175.70 120.71 8.32 4.27(56.41)2.03(29.64) γ 2.34(34.09), Hε 7.56, 6.83 Nε 114.05 H (−4) 173.89 120.718.41 4.67(55.74) 3.26,3.17(29.55) δ 7.23(120.03) ε 8.55(135.92) D (−3)176.03 123.72 8.39 4.61(54.56) 2.70,2.61(41.49) E (−2) 176.19 123.398.52 4.25(57.16) 2.06,1.94(30.38) γ 2.29(36.31) A (−1) 177.93 125.738.27 4.28(53.01) 1.38(19.41) V1 175.77 119.71 7.86 3.83(62.70)1.96(32.84) γCH₃ 0.79(21.09) D2 176.22 124.05 7.97 4.43(54.70)2.46(41.45) N3 175.18 120.71 8.14 4.53(54.10) 2.59(39.05) Hδ 7.49,6.84Hδ 114.20 K4 176.55 121.04 8.20 4.17(57.02) 1.69(32.57) γ 1.25(24.88), δ1.60(29.19), ε 2.94(42.30) F5 176.72 120.38 7.85 5.05(55.43)3.38,3.12(40.06) δ 7.05(131.08) ε 7.05(130.34) ζ 7.27(128.84) N6 175.64122.05 8.43 4.74(52.14) 3.34,2.96(38.29) Hδ 7.48, 6.91 Nδ 111.78 K7178.37 120.38 8.32 4.00(60.16) 1.86(32.41) γ 1.52(24.96), δ 1.71(29.28),ε 3.05(42.46) E8 179.90 121.05 8.21 4.17(59.81) 2.11(29.23) γ2.32(36.72) Q9 177.58 123.05 8.51 3.92(58.92) 2.49(27.33) γ 1.56(33.95),Hε 7.25, 6.95 Nε 112.40 Q10 178.22 120.71 8.75 3.96(59.28) 2.17(28.80) γ2.42(33.87), Hε 7.23, 6.99Nε 113.23 N11 177.32 119.38 8.29 4.62(56.51)2.93(38.45) Hδ 7.73,7.04 Nδ 113.99 A12 178.05 124.05 7.89 4.10(55.68)1.47(18.58) F13 176.01 119.38 8.14 3.80(61.32) 3.32,2.97(39.28) δ 6.93(131.09) ε 7.22(131.92) Y14 178.68 118.37 8.17 3.96(62.41) 3.95(38.48) δ7.15(133.20) ε 6.73(117.95) E15 180.29 120.71 8.54 4.02(60.50)2.15(30.03) γ 2.46(36.87) I16 177.82 121.05 8.41 3.40(65.92) 1.78(37.76)γ 1.78(30.59), γCH₃ 0.76(18.21), δCH₃ 0.53(12.90) L17 176.92 119.04 7.883.70(57.52) 1.13(42.28) γ 1.33(26.62), γCH₃ 0.65(23.99), 0.55(24.91) H18174.46 113.36 7.22 4.52(55.86) 3.47,2.85(29.54) δ 7.09(120.03) ε8.30(135.54) L19 126.06 7.22 4.49(53.55) 1.72,1.38(40.50) γ (26.62),γCH₃ 0.86(23.57), 0.67(27.29) P20 177.98 4.22(65.41) 2.02(32.83) γ2.04(27.59), δ 4.07,3.81(51.79) N21 176.18 115.37 8.88 5.02(52.94)2.91(39.08) Hδ 7.43, 6.87 Nδ 116.28 L22 176.42 119.04 6.49 4.43(54.55)1.69,1.62(43.35) γ 1.69(27.57), γ CH₃ 0.96(24.77), 0.88(22.60) N23175.70 120.71 8.53 4.92(51.58) 3.28,2.84(39.03) Hδ 7.51, 7.43 Nδ 113.50E24 178.14 119.71 8.60 3.96(60.00) 1.97(29.79) γ 2.36(36.18) E25 180.10121.04 8.23 4.07(60.06) 2.07(29.19) γ 2.30(36.80) Q26 178.35 121.38 8.493.99(58.18) 2.48(29.42) γ 2.34(34.26), Hε 8.26, 7.65 Nε 114.39 R27177.75 120.38 8.55 3.79(60.89) 1.74(30.89) γ1.73,1.48(26.92),3.41,3.23(43.15) Hε 7.63 N28 177.64 116.70 8.464.40(56.14) 2.79(38.13) Hδ 7.59, 6.90 Nδ 114.23 A29 180.88 124.72 7.854.18(55.42) 1.34(18.11) F30 177.98 118.37 7.96 4.37(62.55)3.09,2.99(40.05) δ 7.27(131.76) ε 7.12(131.50) I31 177.60 120.04 8.273.79(64.35) 2.11(36.97) γ 1.36(28.96), γCH₃ 0.98(17.98), δCH₃0.63(12.31) Q32 178.23 121.05 8.39 3.96(58.92) 2.22(28.52) γ2.52(34.04), Hδ 7.84, 6.94 Nε 117.88 S33 175.95 116.70 8.06 4.28(62.87)3.99(63.70) L34 177.34 125.73 8.10 3.77(58.02) 1.92(42.63) γ1.64(27.41), δCH₃ 0.78(25.39) K35 178.97 117.04 8.00 4.02(59.81)1.95(32.96) γ 1.62(25.34), δ 1.70(29.84), ε 2.84(42.17) D36 177.29119.38 8.13 4.41(56.88) 2.78,2.71(41.18) D37 115.37 7.57 4.92(51.87)2.58(40.38) P38 178.35 4.50(64.78) 2.23,1.97(32.22) γ 2.24,2.11(27.47),δ 3.87,3.70(50.49) S39 176.14 114.36 8.01 4.34(61.56) 4.05(63.71) Q40176.23 121.72 7.85 4.61(55.31) 2.65(28.41) γ 2.45, 2.32(33.92), Hε 7.59,6.86 Nε 115.77 S41 174.15 117.04 7.77 3.73(63.72) 4.02(62.58) A42 180.94124.72 8.46 4.16(55.83) 1.43(18.38) N43 177.79 120.05 7.89 4.54(56.08)2.89(38.44) Hδ 7.76, 7.00 Nδ 114.50 L44 178.26 123.05 8.58 4.18(58.02)1.79,1.26(42.71) γ 1.87(27.41), δCH₃ 1.11(23.48), 0.78(26.24) L45 177.86120.38 8.41 3.85(58.08) 1.90(42.24) γ 1.53(25.34), δCH₃ 0.90(25.23)) A46181.15 121.05 7.59 4.05(55.51) 1.55(18.28) E47 178.88 120.71 8.054.04(59.25) 2.71(29.77) γ 2.50(35.93) A48 179.50 125.39 8.45 3.48(55.56)0.50(17.74) K49 178.64 119.71 8.48 3.79(60.48) 1.94(32.39) γ1.49(27.13), δ 1.67(30.24), ε 2.89(42.27) K50 179.71 121.38 7.674.11(59.90) 1.96(32.81) γ 1.42(25.18), δ 1.73(29.78), ε 2.98(42.43) L51177.96 123.72 7.90 4.19(57.77) 1.72(42.38) γ 1.57(27.14), δCH₃1.01(25.15) N52 177.46 118.37 8.55 3.97(58.07) 3.11,2.39(42.11) Hδ 7.94,6.85 Nδ 117.79 D53 178.84 120.38 8.23 4.48(57.14) 2.73(40.31) A54 179.27124.39 8.00 4.24(54.56) 1.61(18.81) Q55 174.02 116.37 7.52 4.40(55.23)1.82(28.58) γ 2.65(36.08), Hε 8.74, 7.28 Nε 112.65 A56 126.06 7.104.36(51.14) 1.45(17.92) P57 176.02 4.43(63.35) 2.31,1.97(32.16) γ2.08(27.72), δ 3.79,3.65(50.86) K58 128.74 8.04 4.20(57.41) 1.87(33.81)γ 1,46(24.94), δ 1.68(29.41), ε 3.02(42.11)

[0165] NMR experiments were recorded at a temperature of 25° C. on aVarian Inova 600 spectrometer equipped with a new generation ¹H{¹³C,¹⁵N} triple resonance probe which exhibits a signal-to-noise ratio of1200:1 for a standard 0.1% ethylbenzene sample. At 25° C., thecorrelation time for the overall rotational reorientation of theZ-domain was 4.5 ns (as inferred from measurements of T_(1p)/T₁polypeptide backbone ¹⁵N spin relaxation time ratios (Kay et al.,Biochemistry, 28:8972-8979 (1989); Szyperski et al., J. Biomol. NMR,3:151-164 (1993), which are hereby incorporated by reference in itsentirety)). This value was well within the 3-10 ns range usuallyencountered for medium-sized proteins at ambient temperatures. Hence,the results obtained in the framework of the present study wererepresentative for medium-sized systems in the molecular weight rangefrom about 5 to 20 kDa. NMR spectra were processed and analyzed usingthe programs PROSA (Güntert et al., J. Biomol. NMR, 2:619-629 (1992),which is hereby incorporated by reference in its entirety) and XEASY(Bartels et al., J. Biomol. NMR, 6:1-10 (1995), which is herebyincorporated by reference in its entirety), respectively.

[0166] Specific embodiments of the 8 new RD NMR experiments disclosed bythe present invention as well as 3 other RD NMR experiments that havepreviously been published, were implemented for the present study. FIG.1 provides a survey of (i) the names, (ii) the magnetization transferpathways and (iii) the peak patterns observed in the projected dimensionof each of the 8 RD NMR experiments disclosed by the present inventionas well as 3 other RD NMR experiments that have previously beenpublished. The group comprising the first three experiments are designedto yield “sequential” connectivities via one-bond scalar couplings: 3D H^(α/β) C ^(α/β)(CO)NHN (FIG. 1A; Szyperski et al., J. Magn. Reson., B105: 188-191 (1994), which is hereby incorporated by reference in itsentirety), 3D HACA(CO)NHN (FIG. 1B), and 3D HC(C-TOCSY-CO)NHN (FIG. 1C).The following three experiments provide “intraresidual” connectivitiesvia one-bond scalar couplings: 3D HNNCAHA (FIG. 1D; Szyperski et al., J.Biomol. NMR, 11:387-405 (1998), which is hereby incorporated byreference in its entirety), 3D H ^(α/β) C ^(α/β)COHA (FIG. 1E), and 3D H^(α/β) C ^(α/β)NHN (FIG. 1F). 3D HNN<CO,CA> (FIG. 1G; Szyperski et al.,J. Magn. Reson., B 108: 197-203 (1995); Szyperski et al., J. Am. Chem.Soc., 118:8146-8147 (1996), which are hereby incorporated by referencein their entirety) offers both intraresidual ¹H^(N —) ¹³C^(α) andsequential ¹H^(N)—¹³C′ connectivities. Although 3D HNNCAHA (FIG. 1D), 3DH ^(α/β) C ^(α/β)NHN (FIG. 1F) and 3D HNN<CO,CA> (FIG. 1G) also providesequential connectivities via two-bond ¹³C^(α) _(l−1)—¹⁵N_(l) scalarcouplings, those are usually smaller than the one-bond couplings(Cavanagh et al., Protein NMR Spectroscopy, Academic Press, San Diego,(1996), which is hereby incorporated by reference in its entirety), andobtaining complete backbone resonance assignments critically depends onexperiments designed to provide sequential connectivities via one-bondcouplings (FIGS. 1D-F). 3D HCCH-COSY (FIG. 1H) and 3D HCCH-TOCSY (FIG.1I) allow one to obtain assignments for the “aliphatic” side chain spinsystems, while 2D HBCB(CDCG)HD (FIG. 1J) and 2D ¹H-TOCSY-relayedHCH-COSY (FIG. 1K) provide the corresponding information for the“aromatic” spin systems.

[0167] The RD NMR experiments are grouped accordingly in Table 1, whichlists for each experiment (i) the nuclei for which the chemical shiftsare measured, (ii) if and how the central peaks are acquired and (iii)additional notable technical features. State-of-the art implementations(Cavanagh et al., Protein NMR Spectroscopy, Academic Press, San Diego,(1996); Kay, J. Am. Chem. Soc., 115:2055-2057 (1993); Grzesiek et al.,J. Magn. Reson., 99:201-207 (1992); Montelione et al., J. Am. Chem.Soc., 114:10974-10975 (1992); Boucher et al., J. Biomol. NMR, 2:631-637(1992); Yamazaki et al., J. Am. Chem. Soc., 115:11054-11055 (1993);Zerbe et al., J. Biomol. NMR, 7:99-106 (1996); Grzesiek et al., J.Biomol. NMR, 3:185-204 (1993), which are hereby incorporated byreference in their entirety) making use of pulsed field z-gradients forcoherence selection and/or rejection, and sensitivity enhancement(Cavanagh et al., Protein NMR Spectroscopy, Academic Press, San Diego,(1996), which is hereby incorporated by reference in its entirety) werechosen, which allow executing these experiments with a single transientper acquired free induction decay (FID). Semi (Grzesiek et al., J.Biomol. NMR, 3:185-204 (1993), which is hereby incorporated by referencein its entirety) constant-time (Cavanagh et al., Protein NMRSpectroscopy, Academic Press, San Diego, (1996), which is herebyincorporated by reference in its entirety) chemical shiftfrequency-labeling modules were used throughout in the indirectdimensions in order to minimize losses arising from transverse nuclearspin relaxation. FIGS. 2A-2K provide comprehensive descriptions of theRD NMR r.f. pulse sequences including eight previously unpublished RDNMR r.f. pulse schemes.

[0168] The maximal chemical shift evolution times, which largelydetermine the spectral resolution, as well as the measurement timesinvested for the present study (between 2.7 and 17.1 hours per spectrum)are given in Table 2. The S/N ratio achieved per unit of measurementtime, i.e., the sensitivity, shows only little dependence on therelaxation delay between scans, T_(del), provided that0.7·T₁<T_(del)<1.5·T₁ (Abragam, Principles of Nuclear Magnetism.,Clarendon Press:Oxford (1986); Ernst et al., Principles of NuclearMagnetic Resonance in One and Two Dimensions, Clarendon Press:Oxford(1987), which are hereby incorporated by reference in their entirety).Hence, T_(rel) was set to rather short values around 0.7 seconds.Furthermore, to ensure efficient comparison of peak patterns and shapesmanifested along the projected dimension in the various spectra, the RDNMR experiments in which ¹H and ¹³C are jointly observed in theprojected dimension (“HC”-type experiments; FIG. 1) were acquired withvirtually the same maximal evolution time in t₁(¹³C).

[0169] In total, fourteen RD TR NMR experiments were recorded: 3DHC(C-TOCSY-CO)NHN and 3D HCCH-TOCSY were acquired with two differentmixing times (14 ms and 21 ms) each, and 3D HNNCAHA were acquired withand without adiabatic decoupling of ¹³C^(β) resonances for comparison(Kupce et al., J. Magn. Reson., A 115:273-277 (1995); Matsuo et al., J.Magn. Reson. B 113:190-194 (1996), which are hereby incorporated byreference in their entirety). Except for 3D HNNCAHA , 3D HNN<CO,CA> and2D ¹H-TOCSY-relayed HCH-COSY (FIG. 1), central peaks were derived from¹³C magnetization (FIG. 1; Table 1). Hence, two subspectra, I and IIcontaining the peak pairs and central peaks respectively, were generated(Szyperski et al., J. Am. Chem. Soc., 118:8146-8147 (1996); Szyperski etal., J. Biomol. NMR, 11 :387-405 (1998), which are hereby incorporatedby reference in their entirety) for eight of the RD NMR experiments(FIG. 1). Overall, twenty-four processed RD NMR (sub)spectra were thusobtained for a detailed exploration of relative sensitivities and datacollection strategies. These were complemented with conventional 3DHNNCACB data (Table 2; Wittekind et al., J. Magn. Reson., B 101:201-205(1993), which is hereby incorporated by reference in its entirety).

Example 3 Adjustment of r.f Carrier Frequencies to Minimize SpectralOverlap

[0170] In view of potential peak overlap in spectra recorded for largerproteins, it is of central importance to properly set the r.f. carrierfrequencies. An illustrative example is the 3D HNNCAHA experiment, whereadjustments of the carrier frequencies allows one to place central peaksand upfield and downfield component of the peak pairs into threeseparated spectral regions (Szyperski et al., J. Biomol. NMR, 11:387-405(1998), which is hereby incorporated by reference in its entirety). Thisis accomplished by choosing a ¹H-carrier frequency that yields a minimalin-phase splitting exceeding the ¹³C^(α) chemical shift dispersion(Szyperski et al., J. Biomol. NMR, 11:387-405 (1998), which is herebyincorporated by reference in its entirety). As a consequence, thegeneration of peak pairs does not lead to increased spectral overlap. Infact, the increase in the number of peaks expected for 3D HNNCAHArelative to 3D HNNCA was comparable to the increase observed in widelyused conventional 3D HNNCACB. 3D HNNCACB exhibited up to four peaks foreach amino acid residue: (Wittekind et al., J. Magn. Reson., B101:201-205 (1993), which is hereby incorporated by reference in itsentirety) an intraresidue and a sequential connectivity in each of thequite well separated spectral regions containing the ¹³C^(α) and ¹³C^(β)resonances, respectively. Similarly, 3D HNNCAHA comprised the threeseparated regions each of which may exhibit one intraresidual and onesequential connectivity per amino acid residue (Szyperski et al., J.Biomol. NMR, 11:387-405 (1998), which is hereby incorporated byreference in its entirety).

Example 4 Sensitivity Analysis of RD NMR Experiments

[0171] Since a reduction of dimensionality in a NMR experiment preservesthe relative sensitivity of the higher-dimensional parent experiments,evaluating the relative sensitivity of an entire set of multidimensionalNMR experiments designed to provide complete resonance assignment for aprotein is of general interest. The relative sensitivity of the RD NMRand 3D HNNCACB experiments were analyzed first, by determining the yieldof peak detection, i.e., the ratio of observed peaks over the totalnumber of expected peaks, and second, by separately assessing the S/Nratio distributions of peaks belonging to either RD peak pairs orcentral peaks. Moreover, distinct S/N distributions were then generatedaccording to (i) the atom position involved (e.g., α- or β-moiety in H^(α/β) C ^(α/β)(CO)NHN), (ii) the involvement of intraresidue orsequential connectivities (e.g., ¹³C^(α) _(l)—¹H^(N) _(i) and ¹³ C^(α)_(l−1)—¹H^(N) _(i) connectivities in H ^(α/β) C ^(α/β)NHN) and (iii) theclassification of COSY-type, relay and double-relay peaks in HCCH TOCSY.In total, 127 S/N distributions were thus analyzed (FIG. 5; Table 4).For 3D H ^(α/β) C ^(α/β)(CO)NHN (FIG. 1A) and 3D H ^(α/β) C ^(α/β)COHA(FIG. 1E), there were 4 distributions each: α- and β-connectivities insubspectra I and II. For 3D HACA(CO)NHN (FIG. 1B) and 2D HBCB(CDCG)HD(FIG. 1J), there were 2 distributions each: connectivities in subspectraI and II. For 3D HC(C-TOCSY-CO)NHN (FIG. 1C) recorded with 14 and 21 msmixing time, respectively, there were 10 distributions each: α-, β-, γ-,δ- and ε-connectivities in subspectra I and II. For 3D HNNCAHA (FIG.1D), there were 8 distributions: intraresidual and sequentialconnectivities recorded with and without adiabatic ¹³C^(β) decoupling.For 3D H ^(α/β) C ^(α/β)NHN (FIG. 1F), there were 8 distributions:intraresidual and sequential α- and β-connectivities in subspectra I andII. For 3D HNNCACB, there were 4 distributions: intraresidual andsequential α- and β-connectivities. For 3D HNN<CO,CA> (FIG. 1G), therewere 2 distributions: peak pairs and central peaks. For 3D HCCH-COSY(FIG. 1H), there were 10 distributions: connectivities detected on α-,β-, γ-, δ- and ε-protons for subspectra I and II. For 3D HCCH-TOCSY(FIG. 1H) recorded with 14 and 21 ms mixing time, there were 30distributions each: COSY-type, relay and double-relay connectivitiesdetected on (α-, β-, γ-, δ- and ε-protons for subspectra I and II. For2D ¹H-TOCSY-relayed HCH-COSY (FIG. 1K), there were 3 distributions forconnectivities detected on δ-, ε- and ζ-protons. In order to exclude abias arising from longer transverse relaxation times in several highlydisordered terminal residues (Tashiro et al., J. Mol. Biol., 272:573-590(1997); Lyons et al., Biochemistry, 32:7839-7845 (1993), which arehereby incorporated by reference in their entirety), the N-terminaloctapeptide segment comprising residues “−13” to “−6” (in the numberingchosen in Tashiro et al., J. Mol. Biol., 272:573-590 (1997) and Lyons etal., Biochemistry, 32:7839-7845 (1993), which are hereby incorporated byreference in their entirety) was not considered for the currentsensitivity analyses. To rank the NMR experiments (Table 2) according torelative sensitivity, focus was put on (i) the peak detection yield and(ii) the averaged S/N ratios of those peak categories encoding the primeinformation to be obtained from a given spectrum, i.e., intraresidualconnectivities in HNNCAHA (FIG. 1D), H ^(α/β) C ^(α/β)COHA (FIG. 1E), H^(α/β) C ^(α/β)NHN (FIG. 1F) and HNNCACB, correlation peaks in HCCH-COSYand relay connectivities in HCCH TOCSY. For comparison, these averagedS/N ratios were subsequently divided by the square-root of the NMRmeasurement time (Tables 2 and 4) and scaled relative to the mostsensitive experiment, i.e., HACA(CO)NHN (Table 4; FIG. 5). TABLE 4Signal-to-noise analysis of RD NMR spectra recorded for theZ-domain.^(a) average S/N /{square root}t_(mean) and sensitivity type ofrelative to 3D correla- HACA(CO)NHN RD NMR experiment tion detectionyield average S/N diff 3D HNCAHA D_(i) 60/60(100%)* 13.19 ± 3.66 recorded with adiabatic D_(i-1) 60/54(90%) 4.16 ± 1.76 decoupling of C-βC_(i) 60/60(100%) 8.74 ± 2.99 C_(i-1) 60/45(75%) 3.90 ± 1.92 all240/219(91%) 7.84 3.51/0.25 recorded without adiabatic D_(i) 60/58(97%)7.81 ± 3.45 decoupling of C-β D_(i-1) 60/45(75%) 2.21 ± 1.34 C_(i)60/58(97%) 6.10 ± 3.38 C_(i-1) 60/37(62%) 1.45 ± 1.09 all 240/198(83%)4.85 2.17/0.15 3DH ^(α/β) C ^(α/β)(CO)NHN sub II α 60/60(100%) 13.74 ±4.42  β 60/60(100%) 10.20 ± 5.44  all 122/120(100%)* 11.97  4.81/0.34*sub I α 60/60(100%) 26.41 ± 10.70 β 60/60(100%) 22.29 ± 14.31 all120/120(100%)* 24.35  8.03/0.56* 3D HACA(CO)NHN sub II α 60/60(100%)*27.02 11.62/0.81* sub I α 60/60(100%)* 33.21  14.3/1.00* no central peakacquisition α 60/60(100%) 30.78 18.68/1.30  3D H ^(α/β) C ^(α/β)COHA subII α 60/57(95%) 5.27 ± 2.15 β 60/58(97%) 4.31 ± 1.30 all 120/115(96%)*4.78 1.51/0.11 sub I α 60/60(100%) 10.41 ± 5.31  β 60/60(100%) 9.45 ±7.21 all 60/60(100%)* 9.93 3.14/0.22 3D H ^(α/β) C ^(α/β)NHN sub IIα_(i) 60/60(100%) 8.34 ± 3.84 αi-1 60/46(77%) 3.12 ± 1.87 βi 60/56(93%)3.67 ± 1.59 βi-1 60/60(15%) 2.08 ± 0.55 all 240/171(71%) 3.24 0.78/0.05sub I α_(i) 60/60(100%) 5.93 ± 2.95 α_(i)-1 60/51(85%) 3.08 ± 1.96 β_(i)60/58(97%) 5.35 ± 4.12 β_(i)-1 60/21(35%) 3.55 ± 1.98 all 240/190(79%)4.72 1.14/0.08 3D HNN<CO,CA> CO 60/60(100%)* 47.39 ± 13.44 CA60/60(100%)* 11.28 ± 3.46  all 120/120(100%) 29.34 12.51/0.87  2DHBCB(CGCD)HD sub II δ 7/7(100%)* 10.81  4.70/0.33* sub I δ 7/7(100%)*14.93  6.49/0.45* 2D ¹H-TOCSY-HCH-COSY δ 7/7(100%) 33.64 ± 27.03 ε7/6(86%) 10.75 ± 10.63 ζ 4/3(75%) 6.51 ± 3.54 all 18/16(89%)* 19.9710.83/0.76* 3D HC-(C-TOCSY-CO)NHN 2cyc sub II α 60/60(100%) 8.95 ± 4.98β 60/56(93%) 5.69 ± 4.60 γ 29/15(52%) 2.80 ± 1.51 δ 17/2(12%) 1.40 ±0.24 ε 6/0(0%) all 172/133(77%) 6.77 1.60/0.11 2cyc sub I α 60/60(100%)13.02 ± 7.46  β 60/55(92%) 9.41 ± 9.42 γ 29/24(83%) 4.58 ± 3.46 δ17/8(47%) 2.14 ± 1.05 ε 6/0(0%) all 172/147(85%) 9.70 2.29/0.16 3DHC-(C-TOCSY-CO)NHN 3cyc sub II α 60/58(97%) 5.44 ± 2.84 β 60/42(70%)4.85 ± 3.51 γ 29/17(59%) 2.82 ± 1.02 δ 17/4(24%) 1.27 ± 0.25 e 6/0(0%)all 172/121(70%)* 3.72  0.88/0.06* 3cyc sub I α 60/59(98%) 7.55 ± 4.49 β60/44(73%) 7.34 ± 6.67 γ 29/26(90%) 4.11 ± 3.01 δ 17/14(82%) 2.82 ± 1.55ε 6/4(67%) 1.89 ± 1.17 all 172/147(85%) 6.27 1.48/0.10 HCCH-COSY α74/70(95%) 8.18 ± 8.27 sub II β 98/94(96%) 8.85 ± 5.29 γ 57/54(95%) 8.70± 8.19 δ 22/21(95%) 9.82 ± 10.51 ε 8/8(100%) 18.38 ± 7.76  all259/247(95%)* 9.02  3.62/0.25* HCCH-COSY α 74/70(95%) 9.44 ± 9.60 sub Iβ 98/94(96%) 11.30 ± 9.67  γ 57/54(95%) 11.51 ± 9.02  δ 22/22(100%)20.50 ± 25.50 e 8/8(100%) 29.39 ± 19.61 all 259/248(96%)* 12.22 4.90/0.34* HCCH-TOCSY 2cyc sub I COSY-peaks α 74/68(92%) 5.45 ± 5.22 β98/71(72%) 7.52 ± 6.70 γ 57/50(88%) 5.22 ± 4.67 δ 22/17(77%) 7.21 ± 7.47ε 8/4(50%) 7.82 ± 1.42 all 259/210(81%) 6.28 2.37/0.17 2cyc sub I relaypeaks α 30/21(70%) 3.56 ± 4.96 β 22/18(82%) 4.54 ± 2.25 γ 51/24(47%)6.11 ± 5.36 δ 18/12(67%) 3.44 ± 1.22 ε 4/4(100%) 6.92 ± 4.93 all125/79(63%) 4.23 1.60/0.11 2cyc sub I double relay peaks α 24/5(21%)1.47 ± 1.55 β 8/5(63%) 9.02 ± 5.66 γ 0/0 δ 30/6(20%) 3.86 ± 3.59 ε10/6(60%) 5.97 ± 3.40 all 72/22(31%) 5.62 2.12/0.15 2cyc sub II COSYpeaks α 74/29(39%) β 98/47(48%) γ 57/20(35%) δ 22/9(41%) ε 8/4(50%) all259/105(41%) 2cyc sub II relay peaks α 30/2(0.07%) β 22/5(23%) γ51/7(14% δ 18/0(0%) ε 4/2(50%) all 125/16(13%) 2cyc sub II double relaypeaks α 24/0(0%) β 8/0(0%) γ 0/0(0%) δ 30/0(0%) ε 10/0(0%) all 72(0%)3cyc sub I COSY-peaks α 74/58(78%) 8.91 ± 8.61 β 98/81(83%) 9.18 ± 9.62γ 57/39(68%) 7.79 ± 6.04 δ 22/16(73%) 11.99 ± 10.41 ε 8/4(50%) 16.08 ±2.97  all 259/198(76%) 9.18 3.47/0.25 3cyc sub I relay peaks α30/25(83%) 5.18 ± 5.08 β 22/18(82%) 4.10 ± 2.00 γ 51/26(51%) 5.51 ± 3.09δ 18/13(72%) 5.61 ± 4.56 ε 4/4(100%) 6.82 ± 4.70 all 125/86(69%) 5.201.97/0.14 3cyc sub I double relay peaks α 24/20(83%) 3.34 ± 1.95 β8/4(50%) 15.76 ± 15.28 γ 0/0 δ 30/24(80%) 4.13 ± 1.94 ε 10/10(100%)10.06 ± 6.86  all 72/58(81%)* 7.32  2.77/0.19* 3cyc sub II COSY peaks α174/36(49%) β 98/48(49%) γ 57/19(33%) δ 22/5(23%) ε 8/3(38%) all259/111(43%) 3cyc sub II relay peaks α 30/6(20%) β 22/3(14%) γ51/10(20%) δ 18/0(0%) ε 4/2(50%) all 125/21(17%) 3cyc sub II doublerelay peaks α 20/4(20%) β 8/0(0%) γ 0/0(0%) δ 30/0(0%) ε 4/2(50%) all72/6(0.08%) #(COSY-peaks, relay peaks and double relay peak). The secondcolumn indicates the atom position involved (“type” of correlation), thethird column provides the detection yield (see text and the legend ofFIG. 5), and the fourth column contains the average S/N ratio and thecorresponding standard deviation for all cases where the detection yield(third column) was high. The right-most #column affords the average S/Nratio divided by the square root of the measurement time (Table 2),i.e., the sensitivity. The sensitivity scaled relative to HACA(CO)NHN(number on the right) is also given. Rows labeled with an asterisk (*)contain the values used to create FIG. 5.

[0172] In principle, the relative sensitivities of NMR experiments canbe estimated by calculating transfer amplitudes (Szyperski et al., J.Biomol. NMR, 11:387-405 (1998); Ernst et al., Principles of NuclearMagnetic Resonance in One and Two Dimensions, Clarendon Press:Oxford(1987); Wittekind et al., J. Magn. Reson., B 101:201-205 (1993); Buchleret al., J. Magn. Reson., 125:34-42 (1997), which are hereby incorporatedby reference in their entirety). However, these calculations rely onvarious assumptions such as knowledge about nuclear spin relaxationtimes, or neglect of B₁-inhomogeneity and imperfections of compositepulse decoupling sequences. Hence, an experimental approach is mandatoryto obtain a thorough sensitivity assessment, in particular for theexperiments employed for side chain resonance assignments.

[0173] The key yields of peak detection as well as the relativesensitivity of the NMR spectra recorded for the present study (Tables 1and 2) are shown in FIG. 5. The S/N distribution analysis that wasrequired to generate FIG. 5 is provided in Table 4. Since adiabatic¹³C^(β) decoupling (Kupce et al., J. Magn. Reson., A 115:273-277 (1995);Matsuo et al., J. Magn. Reson. B 113:190-194(1996), which are herebyincorporated by reference in their entirety) increased the sensitivityof 3D HNNCAHA by a factor of about 1.5 (FIG. 5; Table 4), only thedecoupled spectrum was considered in this analysis. Among the group ofexperiments designed to yield sequential connectivities (FIG. 4), all ofthe expected peaks were detected for 3D HACA(CO)NHN (FIGS. 1B and 4) and3D H ^(α/β) C ^(α/β)(CO)NHN (FIGS. 1A and 4). In spite of the ratherlong measurement time of 17 hours (Table 2), a substantial fraction ofthe expected cross peaks was not observed for 3D HC(C-TOCSY-CO)NHN(FIGS. 1C and 4). Evidently, losses due to rotating frame transverserelaxation and off-resonance effects during the C—C TOCSY relay aresignificantly larger than those encountered when implementing the C—CCOSY step which expands 3D HACA(CO)NHN to 3D H ^(α/β) C ^(α/β)(CO)NHN.Moreover, due to the oscillatory nature of the spin modes associatedwith total correlation, (Ernst et al., Principles of Nuclear MagneticResonance in One and Two Dimensions, Clarendon Press:Oxford (1987),which is hereby incorporated by reference in its entirety) the averageS/N ratio observed for a given atom position critically depends on theparticular choice of the mixing time in 3D HC(C-TOCSY-CO)NHN (Table 4):e.g., several β-moiety signals are lost at the expense of detectingadditional γ-, δ- or ε-moiety cross peaks for the long aliphatic sidechains when increasing the mixing time from 14 ms to 21 ms (FIG. 4).

[0174] Among the experiments providing intraresidue connectivities (FIG.6), HNNCAHA (FIGS. 1D and 6A) exhibited complete detection of expectedpeaks and a sensitivity which is comparable to H ^(α/β) C ^(α/β)(CO)NHN,but significantly higher than H ^(α/β) C ^(α/β)COHA (FIGS. 1E and 6B)and H ^(α/β) C ^(α/β)NHN (FIGS. 1F and 6C). The latter experiment,designed in an ‘out-and-stay fashion’ as CBCANHN (Kay, J. Am. Chem.Soc., 115:2055-2057 (1993), which is hereby incorporated by reference inits entirety), is the least sensitive among the suite of RD NMRexperiments studied here and can thus be expected to be primarily of usefor smaller proteins. However, virtually all expected correlations wereobserved. Conventional HNNCACB is slightly more sensitive than HNNCAHAand equally sensitive as H ^(α/β) C ^(α/β)(CO)NHN. However, whenconsidering symmetrization of [ω₁(¹³C),ω₃(¹H^(N))]-strips about centralpeaks along ω₁, (Szyperski et al., J. Magn. Reson., B 108: 197-203(1995); Szyperski et al., J. Biomol. NMR, 11:387-405 (1998), which arehereby incorporated by reference in their entirety) HNNCAHA can beconsidered to be more sensitive than HNNCACB even for smaller proteins.HNN<CO,CA> (FIG. 1G) offers both intraresidue ¹H^(N)—¹³C^(α) (peakpairs) and sequential ¹H^(N)—¹³C′ (central peaks) connectivities. Inaccordance with the outstanding sensitivity of HNNCO, central peakdetection in HNN<CO,CA> was by far the most sensitive observed in allspectra, while the sensitivity of corresponding peak pair detection wascomparable to HNNCAHA . Hence, central peaks in 3D HNN<CO,CA> may berecruited for secure spin system identification (Zimmerman et al., J.Mol. Biol., 269:592-610 (1997), which is hereby incorporated byreference in its entirety) in cases of overlap in 2D [¹⁵N, ¹H]-HSQC.

[0175] The sensitivity of peak pair detection in 3D HCCH COSY, requiredfor aliphatic side chain assignment, was again comparable to 3D HNNCAHA, while detection of relayed COSY peaks in 3D HCCH TOCSY was slightlyless sensitive. The incompleteness of relay peak detection was, however,to some extent due to signal overlap (Table 4). 2D HBCB(CDCG)HD and 2D¹H-TOCSY-relayed HCH-COSY, providing the aromatic spin systemassignments, appeared to be rather sensitive. However, analysis for theZ-domain was biased by (i) the relatively small number of aromaticresidues, and (ii) their partly flexibly disordered nature (His(—4), Phe5 and Phe 13 exhibit local displacements that are well above the averagefor residues buried in the molecular core; protein data bank accessioncode: 2SPZ). When involving only those aromatic rings that areapparently not flexibly disordered, 2D HBCB(CDCG)HD appeared to beslightly less sensitive than 3D HCCH COSY.

[0176] Overall (FIG. 5), (i) outstanding sensitivity was found for 3DHACA(CO)NHN, (ii) similar sensitivity was found for 3D H ^(α/β) C^(α/β)(CO)NHN, 3D HNNCAHA , 3D HNN<CO,CA>, 3D HNNCACB, 3D HCCH COSY and2D ¹H-TOCSY-relayed HCH-COSY, (iii) slightly reduced sensitivity wasfound for 3D H ^(α/β) C ^(α/β)COHA, 2D HBCB(CDCG)HD and relay peakdetection in 3D HCCH TOCSY, and (iv) the lowest sensitivity was foundfor 3D HC(C-TOCSY-CO)NHN and 3D H ^(α/β) C ^(α/β)NHN. In the “H ^(α/β) C^(α/β)-experiments, the averaged intensity of the α- and β-moiety peakpairs was quite similar (though the S/N distribution of the β-peaks wasbroader reflecting larger variations in transverse relaxation times),and the central peaks exhibited a sensitivity of about two thirdsrelative to the individual peaks of the peak pairs. However, since thenon-selective ¹³C T₁-relaxation times are shorter than the ¹H T₁-timesat higher molecular weight (Abragam, Principles of Nuclear Magnetism.,Clarendon Press:Oxford (1986); Ernst et al., Principles of NuclearMagnetic Resonance in One and Two Dimensions, Clarendon Press:Oxford(1987), which are hereby incorporated by reference in their entirety),the relative sensitivity of central peak detection using¹³C-magnetization becomes more favorable for larger systems. Moreover,the relative sensitivity of the various experiments shifts relative toeach other with increasing molecular weight (Buchler et al., J. Magn.Reson., 125:34-42 (1997), which is hereby incorporated by reference inits entirety). In particular, 3D HNNCACB and 3D H ^(α/β) C ^(α/β)COHAcan be expected to loose relative sensitivity for larger systems sincetransverse magnetization resides comparably long on rapidly relaxing¹³C^(α).

Example 5 HTP Assignment Strategy: A “Standard Set” of RD NMRExperiments

[0177] The comprehensive analysis of the suite of multidimensionalspectra recorded for the present study (FIG. 5; Tables 1 and 2) lays thefoundation to devise strategies for RD NMR-based HTP resonanceassignment of proteins.

[0178] For proteins in the molecular weight range up to about 20 kDa, 3DH ^(α/β) C ^(α/β)(CO)NHN plays a pivotal role (FIG. 7). Firstly, thepeak patterns observed along ω₁(¹³C^(α/β)) in subspectra I and II enablesequential resonance assignment in combination with HNNCAHA and HNNCACB,respectively, by matching intraresidue and sequential ¹H^(α), ¹³C^(α)and ¹³C^(β) chemical shifts (FIG. 8). (When considering ‘nuclear spinrelaxation time labeling’ of peak pairs, subspectrum II derived from ¹³Csteady state magnetization provides largely redundant information whencompared with subspectrum I. However, the observation of the centralpeaks allows direct matching of peak positions between subspectrum II,essentially a CBCA(CO)NHN spectrum, and HNNCACB (FIG. 6).) Moreover,this set of chemical shifts alone provides valuable information foramino acid type identification (Zimmerman et al., J. Mol. Biol.,269:592-610 (1997); Cavanagh et al., Protein NMR Spectroscopy, AcademicPress, San Diego, (1996); Grzesiek et al., J. Biomol. NMR, 3:185-204(1993), which are hereby incorporated by reference in their entirety).Complementary recording of 3D H ^(α/β) C ^(α/β)COHA and 3D HNN<CO,CA>contributes polypeptide backbone ¹³C═O chemical shift measurements forestablishing sequential assignments: the intraresidue correlation isobtained by ω₁(¹³C^(α/β)) peak pattern matching (FIGS. 9A-B) with 3D H^(α/β) C ^(α/β)(CO)NHN, and the sequential correlation is inferred from¹³C^(α), ¹⁵N and ¹H^(N) chemical shifts in 3D HNN<CO,CA> (Szyperski etal., J. Biomol. NMR, 11:387-405 (1998), which is hereby incorporated byreference in its entirety). Notably, even for medium-sized(non-deuterated) proteins this approach is superior to the use of a lowsensitivity HNNCACO-type experiment (e.g., in combination with HNNCOCA),where the magnetization transfer via rapidly relaxing ¹³C^(α) relies onthe rather small ¹⁵N—¹³C^(α) one-bond scalar coupling. Secondly,comparison of ω₁(¹³C^(α/β)) peak patterns with 3D HCCH-COSY (FIG. 10)and TOCSY connects the C^(α/β)/H^(α/β) chemical shifts with those of thealiphatic side chain spin systems (For Z-domain, complete side chainassignments were obtained for all but six residues using 3D HCCH-COSYonly.) (FIGS. 10 and 11), while comparison of ω₁(¹³C^(β)) peaks with 2DHBCB(CDCG)HD and subsequent linking with ¹H^(δ) chemical shifts detectedin 2D ¹H-TOCSY-relayed HCH-COSY affords assignment of the aromatic spinsystems (FIG. 12). Since for many amino acid residues the two β-protonsexhibit non-degenerate chemical shifts, the connection of H ^(α/β) C^(α/β)(CO)NHN and HBCB(CDCG)HD or HCCH-COSY/TOCSY (FIG. 7) may in factoften rely on comparison of three chemical shifts, i.e., δ(¹H^(β2)),δ(¹H^(β3)) and δ(¹³C^(β)). This consideration underscores the potentialof recruiting β-proton chemical shifts for establishing sequentialresonance assignments.

[0179] The ‘standard set’ of nine experiments (labeled with asterisks inTable 2) as described in the above paragraph required 60 hours ofinstrument time for the Z-domain on our 600 MHz NMR system (Table 2).However, the minimal S/N ratios detected (Table 4) reveal that half ofthe measurement time would have been sufficient for backbone amideproton detected experiments, indicating that these spectra were stillacquired in the sampling limited regime. (The lowest S/N peak ratios arearound 5:1, which implies that a reduction of by could be afforded. Afurther indication of an inappropriately long measurement time is due tothe fact that nearly all sequential connectivities relying on two-bondscalar couplings (Güntert et al., J. Biomol. NMR, 2:619-629 (1992),which is hereby incorporated by reference in its entirety) were observedin 3D HNNCAHA (FIG. 7): nearly all ¹H^(N), ¹⁵N, ¹³C^(α) and ¹H^(α)backbone resonances of Z-domain could be assigned using this spectrum(Szyperski et al., J. Biomol. NMR, 11:387-405 (1998), which is herebyincorporated by reference in its entirety) alone.) Hence, a nearlycomplete resonance assignment of the Z-domain could have been obtainedfrom the standard set in about 40 hours, if the RD backbone experimentswere conducted with a single transient per acquired FID. (The suite ofexperiments in Table 1 may provide complete resonance assignments ofproteins, excluding only the side chain NH_(n) moieties, the CH^(ε)groups of histidinyl, and the CH^(ε3), CH^(ζ2,3) and CH^(η2) groups oftryptophanyl residues, which can be obtained as described in Yamazaki etal., J. Am. Chem. Soc., 115:11054-11055 (1993), which is herebyincorporated by reference in its entirety. Notably, the protein studiedhere does not contain tryptophan residues.) This outstandingly shortmeasurement time needs to be compared with 1-3 weeks of measurement timethat are currently routinely invested to assign medium-sized proteins.Concomitantly, the high redundancy for establishing sequentialconnectivities using this suite of experiments (six projected 4D, one 3Dand two projected 3D experiments) greatly supports robust automatedassignment. Importantly, the information encoded in each projected 4Dspectrum cannot be obtained by simply recording two 3D spectra: in casesof chemical shift degeneracy a chemical shift quartuple is notequivalent to two shift triples.

Example 6 Sensitivity Profile Within the “Standard Set” of NMRExperiments

[0180] It is desirable that the NMR experiments applied for proteinresonance assignment in a high-throughput manner exhibit comparablesensitivity. This is because the prediction of the totally requiredmeasurement times is facilitated (roughly a multiple of the measurementtime of an arbitrarily chosen single experiment) and the signal-to-noiseratios observed in the experiment conducted first allow one to readilyadjust the (rather similar) measurement times of the remaining oneswhile the recording of the set of experiments is in progress. It is thusimportant to note that the sensitivity within the standard set of nineexperiments (Table 2) varies by only about a factor of two whencomparing peak pair detection in 3D H ^(α/β) C ^(α/β)(CO)NHN with relayCOSY peak detection in 3D HCCH-TOCSY (FIG. 5). Extraordinarily sensitivecentral peak detection in 3D HNN<CO,CA> represents the sole exception.However, the availability of extremely sensitive detection of (¹H^(N),¹⁵N, ¹³C═O) chemical shift triples is of high value for identificationof spin systems (Zimmerman et al., J. Mol. Biol., 269:592-610 (1997),which is hereby incorporated by reference in its entirety). In fact,this apparent exception thus neatly complements the even sensitivityprofile of the remaining experiments.

Example 7 A “Minimal Set” of RD NMR Experiments

[0181] For Z-domain, six RD NMR experiments were actually sufficient toprovide the desired resonance assignments: 3D H ^(α/β) C ^(α/β)(CO)NHN,3D HNNCAHA , 3D HCCH-COSY/TOCSY, 2D HBCB(CDCG)HD and 2D ¹H-TOCSY-relayedHCH-COSY. This set of experiments was recorded within 36 hours ofinstrument time (Table 2), and can be considered as a ‘minimal set’ ofRD NMR experiments for HTP resonance assignment of proteins up to around10 kDa. For smaller proteins, the use of 3D HC(C-TOCSY-CO)NHN, 3D H^(α/β) C ^(α/β)NHN, 3D HCCH-COSY, 2D HBCB(CDCG)HD and 2D¹H-TOCSY-relayed HCH-COSY represents a viable alternative to rapidlyobtain assignments (Table 1).

[0182] Although preferred embodiments have been depicted and describedin detail herein, it will be apparent to those skilled in the relevantart that various modifications, additions, substitutions, and the likecan be made without departing from the spirit of the invention and theseare therefore considered to be within the scope of the invention asdefined in the claims which follow.

What is claimed:
 1. A method of conducting a reduced dimensionalitythree-dimensional (3D) HA,CA,(CO),N,HN nuclear magnetic resonance (NMR)experiment by measuring the chemical shift values for the followingnuclei of a protein molecule having two consecutive amino acid residues,i−1 and i: (1) an α-proton of amino acid residue i−1, ¹H^(α) _(i−1); (2)an (α-carbon of amino acid residue i−1, ¹³C^(α) _(l−1); (3) apolypeptide backbone amide nitrogen of amino acid residue i, ¹⁵N_(i);and (4) a polypeptide backbone amide proton of amino acid residue i,¹H^(N) _(i), said method comprising: providing a protein sample;applying radiofrequency pulses to the protein sample which effect anuclear spin polarization transfer wherein the chemical shift evolutionsof ¹H^(α) _(l−1) and ¹³C^(α) _(l−1) of amino acid residue i−1 areconnected to the chemical shift evolutions of ¹⁵N_(i) and ¹H^(N) _(i) ofamino acid residue i, under conditions effective (1) to generate NMRsignals encoding the chemical shift values of ¹³C^(α) _(l−1) and ¹⁵N_(l)in a phase sensitive manner in two indirect time domain dimensions,t₁(¹³C^(α)) and t₂(¹⁵N), respectively, and the chemical shift value of¹H^(N) _(i) in a direct time domain dimension, t₃(¹H^(N)), and (2) tocosine modulate the ¹³C^(α) _(l−1) chemical shift evolution int₁(¹³C^(α)) with the chemical shift evolution of ¹H^(α) _(l−1); andprocessing the NMR signals to generate a 3D NMR spectrum with a primarypeak pair derived from said cosine modulating, wherein (1) the chemicalshift values of ¹⁵N_(i) and ¹H^(N) _(i) are measured in two frequencydomain dimensions, ω₂( ⁵N) and ω₃(¹H^(N)), respectively, and (2) thechemical shift values of ¹H^(α) _(i−1) and ¹³C^(α) _(i−1) are measuredin a frequency domain dimension, ω₁(¹³C^(α)), by the frequencydifference between the two peaks forming said primary peak pair and thefrequency at the center of the two peaks, respectively.
 2. The methodaccording to claim 1, wherein said applying radiofrequency pulses iscarried out so that the chemical shift evolution of ¹⁵N_(l) does notoccur and said processing the NMR signals generates a two dimensional(2D) NMR spectrum with a peak pair wherein (1) the chemical shift valueof ¹H^(N) _(i) is measured in a frequency domain dimension, ω₂(¹H^(N)),and (2) the chemical shift values of ¹H^(α) _(i−1) and ¹³C^(α) _(i−1)are measured in a frequency domain dimension, ω₁(¹³C^(α)), by thefrequency difference between the two peaks forming said primary peakpair and the frequency at the center of the two peaks, respectively. 3.The method according to claim 1, wherein said applying radiofrequencypulses is carried out so that the chemical shift evolution of apolypeptide backbone carbonyl carbon of amino acid residue i−1,¹³C′_(l−1), occurs under conditions effective to generate NMR signalsencoding the chemical shift value of ¹³C′_(i−1) in a phase sensitivemanner in an indirect time domain dimension, t₄(¹³C′), and saidprocessing the NMR signals generates a four dimensional (4D) NMRspectrum with a peak pair wherein (1) the chemical shift values of¹⁵N_(l), ¹H^(N) _(i) and ¹³C′_(l−1) are measured in three frequencydomain dimensions, ω₂(¹⁵N), ω₃(¹H^(N)), and ω₄(¹³C′), respectively, and(2) the chemical shift values of ¹H^(α) _(i−1) and ¹³C^(α) _(i−1) aremeasured in a frequency domain dimension, ω₁(¹³C^(α)), by the frequencydifference between the two peaks forming said peak pair and thefrequency at the center of the two peaks, respectively.
 4. The methodaccording to claim 1, wherein said applying radiofrequency pulses iscarried out under conditions effective to additionally cosine modulatethe ¹³C^(α) _(l−1) chemical shift evolution in t₁(¹³C^(α)) with thechemical shift evolution of a polypeptide backbone carbonyl carbon ofamino acid residue i−1, ¹³C′_(l−1), and said processing the NMR signalsgenerates a 3D NMR spectrum with two secondary peak pairs wherein (1)each of the secondary peak pairs is derived from a different one of thepeaks of the primary peak pair, and (2) the chemical shift value of¹³C′_(l−1) is measured along ω₁(¹³C^(α)) by the frequency differencebetween the two peaks forming one of the secondary peak pairs.
 5. Themethod according to claim 4, wherein said applying radiofrequency pulsesis carried out under conditions effective (1) to generate an additionalNMR signal encoding the chemical shift values of ¹³C^(α) _(i−1) and¹⁵N_(i) in a phase sensitive manner in t₁(¹³C^(α)) and t₂(¹⁵N) and thechemical shift value of ¹H^(N) _(i) in t₃(¹H^(N)), (2) to cosinemodulate the ¹³C^(α) _(i−1) chemical shift evolution in t₁(¹³C^(α)) withthe chemical shift evolution of ¹³C′_(l−1), and (3) to avoid cosinemodulating the ¹³C^(α) _(l−1) chemical shift evolution in t₁(¹³C^(α))with the chemical shift evolution of ¹H^(α) _(i−1), and said processingthe NMR signals and the additional NMR signal generates a 3D NMRspectrum with an additional secondary peak pair located between said twosecondary peak pairs which measures the chemical shift values of¹³C′_(l−1) and ¹³C^(α) _(i−1) along ω₁(¹³C^(α)), by the frequencydifference between the two peaks forming the additional secondary peakpair and the frequency at the center of said two peaks, respectively. 6.The method according to claim 5, wherein said additional secondary peakpair is derived from ¹³C^(α) nuclear spin polarization.
 7. The methodaccording to claim 6, wherein said applying radiofrequency pulseseffects a nuclear spin polarization transfer according to FIG. 1B,wherein a radiofrequency pulse is used to create transverse ¹H^(α)_(i−1) magnetization, and ¹H^(α) _(i−1) magnetization is transferred to¹³C^(α) _(l−1), to ¹⁵N_(l), and to ¹H^(N) _(i), where the NMR signal isdetected.
 8. The method according to claim 7, wherein said applyingradiofrequency pulses comprises: applying a first set of radiofrequencypulses according to the scheme shown in FIG. 2B to generate a first NMRsignal, and applying a second set of radiofrequency pulses according tothe scheme shown in FIG. 2B, wherein phase φ₁ of the first ¹H pulse isaltered by 180° to generate a second NMR signal, said method furthercomprising: adding and subtracting the first NMR signal and the secondNMR signal prior to said processing, whereby said processing the NMRsignals generates a first NMR subspectrum derived from said subtractingwhich contains said two secondary peak pairs, and a second NMRsubspectrum derived from said adding which contains said additionalsecondary peak pair.
 9. The method according to claim 1, wherein saidapplying radiofrequency pulses is carried out under conditions effective(1) to generate an additional NMR signal encoding the chemical shiftvalues of ³C^(α) _(i−1) and ¹⁵N_(i) in a phase sensitive manner int₁(¹³C^(α)) and t₂(¹⁵N) and the chemical shift value of ¹H^(N) _(i) int₃(¹H^(N)), and (2) to avoid cosine modulating the ¹³C^(α) _(i−1)chemical shift evolution in t₁(¹³C^(α)) with the chemical shiftevolution of ¹H^(α) _(i−1) for the additional NMR signal, and saidprocessing the NMR signals and the additional NMR signal generates a 3DNMR spectrum with an additional peak located centrally between two peaksforming said primary peak pair which measures the chemical shift valueof ¹³C^(α) _(i−1) along ω₁(¹³C^(α)).
 10. The method according to claim9, wherein said additional peak is derived from ¹³C^(α) nuclear spinpolarization.
 11. The method according to claim 10, wherein saidapplying radiofrequency pulses effects a nuclear spin polarizationtransfer according to FIG. 1B, wherein a radiofrequency pulse is used tocreate transverse ¹H^(α) _(i−1) magnetization, which is transferred to¹³C^(α) _(i−1), to ¹⁵N_(i), and to ¹H^(N) _(i), to generate the NMRsignal.
 12. The method according to claim 11, wherein said applyingradiofrequency pulses comprises: applying a first set of radiofrequencypulses according to the scheme shown in FIG. 2B to generate a first NMRsignal, and applying a second set of radiofrequency pulses according tothe scheme shown in FIG. 2B, wherein phase φ₁ of the first ¹H pulse isaltered by 180° to generate a second NMR signal, said method furthercomprising: adding and subtracting the first NMR signal and the secondNMR signal prior to said processing, whereby said processing the NMRsignals generates a first NMR subspectrum derived from said subtractingwhich contains said primary peak pair and a second NMR subspectrumderived from said adding which contains said additional peak locatedcentrally between the two peaks forming said primary peak pair.
 13. Amethod of conducting a reduced dimensionality three-dimensional (3D)H,C,—(C-TOCSY-CO),N,HN nuclear magnetic resonance (NMR) experiment bymeasuring the chemical shift values for the following nuclei of aprotein molecule having two consecutive amino acid residues, i−1 and i:(1) aliphatic protons of amino acid residue i−1, ¹H^(ali) _(i−1); (2)aliphatic carbons of amino acid residue i−1, ¹³C^(ali) _(i−1); (3) apolypeptide backbone amide nitrogen of amino acid residue i, ¹⁵N_(l);and (4) a polypeptide backbone amide proton of amino acid residue i,¹H^(N) _(i), said method comprising: providing a protein sample;applying radiofrequency pulses to the protein sample which effect anuclear spin polarization transfer wherein the chemical shift evolutionsof ¹H^(ali) _(i−1) and ¹³C^(ali) _(i−1) of amino acid residue i−1 areconnected to the chemical shift evolutions of ¹⁵N_(i) and ¹H^(N) _(i) ofamino acid residue i, under conditions effective (1) to generate a NMRsignal encoding the chemical shifts of ¹³C^(ali) _(i−1) and ¹⁵N_(i) in aphase sensitive manner in two indirect time domain dimensions,t₁(¹³C^(ali)) and t₂(¹⁵N), respectively, and the chemical shift of¹H^(N) _(i) in a direct time domain dimension, t₃(¹H^(N)), and (2) tocosine modulate the chemical shift evolutions of ¹³C^(ali) _(i−1) int₁(¹³C^(ali)) with the chemical shift evolutions of ¹H^(ali) _(i−1); andprocessing the NMR signals to generate a 3D NMR spectrum with peak pairsderived from said cosine modulating wherein (1) the chemical shiftvalues of ¹⁵N_(i) and ¹H^(N) _(i) are measured in two frequency domaindimensions, ω₂(¹⁵N) and ω₃(¹H^(N)), respectively, and (2) the chemicalshift values of ¹H^(ali) _(i−1) and ¹³C^(ali) _(i−1) are measured in afrequency domain dimension, ω₁(¹³C^(ali)), by the frequency differencesbetween the two peaks forming said peak pairs and the frequencies at thecenter of the two peaks, respectively.
 14. The method according to claim13, wherein said applying radiofrequency pulses is carried out so thatthe chemical shift evolution of ¹⁵N_(l) does not occur and saidprocessing the NMR signals generates a two dimensional (2D) NMR spectrumwith peak pairs wherein (1) the chemical shift value of ¹H^(N) _(i) ismeasured in a frequency domain dimension, ω₂(¹H^(N)), and (2) thechemical shift values of ¹H^(ali) _(l−1) and ¹³C^(ali) _(i−1) aremeasured in a frequency domain dimension, ω₁(¹³C^(ali)), by thefrequency differences between the two peaks forming said peak pairs andthe frequencies at the center of the two peaks, respectively.
 15. Themethod according to claim 13, wherein said applying radiofrequencypulses is carried out so that the chemical shift evolution of apolypeptide backbone carbonyl carbon of amino acid residue i−1,¹³C′_(l−1), occurs under conditions effective to generate NMR signalsencoding the chemical shift value of ¹³C′_(i−1) in a phase sensitivemanner in an indirect time domain dimension, t₄(¹³C′), and saidprocessing the NMR signals generates a four dimensional (4D) NMRspectrum with variant peak pairs wherein (1) the chemical shift valuesof ¹⁵N_(i), ¹H^(N) _(l) and ¹³C′_(i−1) are measured in three frequencydomain dimensions, ω₂(¹⁵N), ω₃(¹H^(N)), and ω₄(¹³C′), respectively, and(2) the chemical shift values of ¹H^(ali) _(i−1) and ¹³C^(ali) _(i−1)are measured in a frequency domain dimension, ω₁(¹³C^(ali)), by thefrequency differences between the two peaks forming said variant peakpairs and the frequencies at the center of the two peaks, respectively.16. The method according to claim 13, wherein said applyingradiofrequency pulses is carried out under conditions effective (1) togenerate an additional NMR signal encoding the chemical shift values of¹³C^(ali) _(i−1) and ¹⁵N_(i) in a phase sensitive manner int₁(¹³C^(ali)) and t₂(¹⁵N) and the chemical shift value of ¹H^(N) _(i) int₃(¹H^(N)), and (2) to avoid cosine modulating the chemical shiftevolutions of ¹³C^(ali) _(l−1) in t₁(¹³C^(ali)) with the chemical shiftevolution of ¹H^(α) _(l−1) for the additional NMR signal, and saidprocessing the NMR signals and the additional NMR signal generates a 3DNMR spectrum with additional peaks located centrally between said peakpairs which measure the chemical shift values of ¹³C^(ali) _(l−1) alongω₁(¹³C^(ali)).
 17. The method according to claim 16, wherein saidadditional peaks are derived from ¹³C^(ali) nuclear spin polarization.18. The method according to claim 17, wherein said applyingradiofrequency pulses effects a nuclear spin polarization transferaccording to FIG. 1C, wherein a radiofrequency pulse is used to createtransverse ¹H^(ali) _(i−1) magnetization, and ¹H^(ali) _(i−1)magnetization is transferred to ¹³C^(ali) _(l−1), to ¹³C^(α) _(i−1), to¹³C′_(l−1), to ¹⁵N_(i), and to ¹H^(N) _(l), where the NMR signal isdetected.
 19. The method according to claim 18, wherein said applyingradiofrequency pulses comprises: applying a first set of radiofrequencypulses according to the scheme shown in FIG. 2C to generate a first NMRsignal, and applying a second set of radiofrequency pulses according tothe scheme shown in FIG. 2C, wherein phase φ₁ of the first ¹H pulse isaltered by 180° to generate a second NMR signal, said method furthercomprising: adding and subtracting the first NMR signal and the secondNMR signal prior to said processing, whereby said processing the NMRsignals generates a first NMR subspectrum derived from said subtractingwhich contains said peak pairs, and a second NMR subspectrum derivedfrom said adding which contains said additional peaks located centrallybetween said peak pairs.
 20. A method of conducting a reduceddimensionality three-dimensional (3D) H ^(α/β),C ^(α/β),CO,HA nuclearmagnetic resonance (NMR) experiment by measuring the chemical shiftvalues for the following nuclei of a protein molecule having an aminoacid residue, i: (1) a β-proton of amino acid residue i, ¹H^(β) _(l);(2) a β-carbon of amino acid residue i, ¹³C^(β) _(i); (3) an α-proton ofamino acid residue i, ¹H^(α) _(i); (4) an α-carbon of amino acid residuei, ¹³C^(α) _(i); and (5) a polypeptide backbone carbonyl carbon of aminoacid residue i, ¹³C′_(l), said method comprising: providing a proteinsample; applying radiofrequency pulses to the protein sample whicheffect a nuclear spin polarization transfer wherein the chemical shiftevolutions of ¹H^(α) _(i), ¹H^(β) _(i), ¹³C^(α) _(i), and ¹³C^(β) _(i)are connected to the chemical shift evolution of ¹³C′_(i), underconditions effective (1) to generate NMR signals encoding the chemicalshift values of ¹³C^(α) _(i), ¹³C^(β) _(i) and ¹³C′_(l) in a phasesensitive manner in two indirect time domain dimensions, t₁(¹³C^(α/β))and t₂(¹³C′), respectively, and the chemical shift value of ¹H^(α) _(i)in a direct time domain dimension, t₃(¹H^(α)), and (2) to cosinemodulate the chemical shift evolutions of ¹³C^(α) _(i) and ¹³C^(β) _(l)in t₁(¹³C^(α/β)) with the chemical shift evolutions of ¹H^(α) _(i) and¹H^(β) _(i), respectively; and processing the NMR signals to generate a3D NMR spectrum with peak pairs derived from said cosine modulatingwherein (1) the chemical shift values of ¹³C′_(i) and ¹H^(α) _(i) aremeasured in two frequency domain dimensions, ω₂(¹³C′) and ω₃(¹H^(α)),respectively, and (2) (i) the chemical shift values of ¹H^(α) _(l) and¹H^(β) _(l) are measured in a frequency domain dimension, ω₁(¹³C^(α/β)),by the frequency differences between the two peaks forming said peakpairs, and (ii) the chemical shift values of ¹³C^(α) _(i), and ¹³C^(β)_(l) are measured in a frequency domain dimension, ω₁(¹³C^(α/β)), by thefrequencies at the center of the two peaks forming said peak pairs. 21.The method according to claim 20, wherein said applying radiofrequencypulses is carried out so that the chemical shift evolution of ¹³C′_(l)does not occur and said processing the NMR signals generates a twodimensional (2D) NMR spectrum with peak pairs wherein (1) the chemicalshift value of ¹H^(α) _(i) is measured in a frequency domain dimension,ω₂(¹H^(α)), and (2) (i) the chemical shift values of ¹H^(α) _(i) and¹H^(β) _(l) are measured in a frequency domain dimension, ω₁(¹³C^(α/β)),by the frequency differences between two peaks forming said peak pairs,respectively, and (ii) the chemical shift values of ¹³C^(α) _(i), and¹³C^(β) _(i) are measured in a frequency domain dimension,ω₁(¹³C^(α/β)), by the frequencies at the center of the two peaks formingsaid peak pairs.
 22. The method according to claim 20 wherein saidapplying radiofrequency pulses is carried out under conditions effective(1) to generate an additional NMR signal encoding the chemical shiftvalues of ¹³C^(α) _(i), ¹³C^(β) _(i) and ¹⁵N_(i) in a phase sensitivemanner in t₁(¹³C^(α/β)) and t₂(¹⁵N) and the chemical shift value of¹H^(α) _(i) in t₃(¹H^(α)), and (2) to avoid cosine modulating thechemical shift evolutions of ¹³C^(α) _(i) and ¹³C^(β) _(i) int₁(¹³C^(α/β)) with the chemical shift evolutions of ¹H^(α) _(l) and¹H^(β) _(l) for the additional NMR signal, and said processing the NMRsignals and the additional NMR signal generates a 3D NMR spectrum withadditional peaks located centrally between the two peaks forming saidpeak pairs which measure the chemical shift values of ¹³C^(α) _(i) and¹³C^(β) _(i) along ω₁(¹³C^(α/β)).
 23. The method according to claim 22,wherein said additional peaks are derived from ¹³C^(α) and ¹³C^(β)nuclear spin polarization.
 24. The method according to claim 23, whereinsaid applying radiofrequency pulses effects a nuclear spin polarizationtransfer according to FIG. 1E, wherein a radiofrequency pulse is used tocreate transverse ¹H^(α) _(i) and ¹H^(β) _(l) magnetization, and ¹H^(α)_(l) and ¹H^(β) _(i) polarization is transferred to ¹³C^(α) _(i) and¹³C^(β) _(i), to ¹³C′_(i), and back to ¹H^(α) _(i), where the NMR signalis detected.
 25. The method according to claim 24, wherein said applyingradiofrequency pulses comprises: applying a first set of radiofrequencypulses according to the scheme shown in FIG. 2E to generate a first NMRsignal, and applying a second set of radiofrequency pulses according tothe scheme shown in FIG. 2E, wherein phase φ₁ of the first ¹H pulse isaltered by 180° a to generate a second NMR signal, said method furthercomprising: adding and subtracting the first NMR signal and the secondNMR signal prior to said processing, whereby said processing the NMRsignals generates a first NMR subspectrum derived from said subtractingwhich contains said peak pairs, and a second NMR subspectrum derivedfrom said adding which contains said additional peaks located centrallybetween the two peaks forming said peak pairs.
 26. A method ofconducting a reduced dimensionality three-dimensional (3D) H ^(α/β),C^(α/β),N,HN nuclear magnetic resonance (NMR) experiment by measuring thechemical shift values for the following nuclei of a protein moleculehaving an amino acid residue, i: (1) a β-proton of amino acid residue i,¹H^(β) _(l); (2) a β-carbon of amino acid residue i, ¹³C^(β) _(i); (3)an α-proton of amino acid residue i, ¹H^(α); (4) an α-carbon of aminoacid residue i, ¹³C^(α) _(i); (5) a polypeptide backbone amide nitrogenof amino acid residue i, ¹⁵N_(i); and (6) a polypeptide backbone amideproton of amino acid residue i, ¹H^(N) _(l), said method comprising:providing a protein sample; applying radiofrequency pulses to theprotein sample which effect a nuclear spin polarization transfer whereinthe chemical shift evolutions of ¹H^(α) _(i), ¹H^(β) _(l), ¹³C^(α) _(l),and ¹³C^(β) _(i) are connected to the chemical shift evolutions of¹⁵N_(i) and ¹H^(N) _(i), under conditions effective (1) to generate NMRsignals encoding the chemical shift values of ¹³C^(α) _(i), ¹³C^(β) _(i)and ¹⁵N_(l) in a phase sensitive manner in two indirect time domaindimensions, t₁(¹³C^(α/β)) and t₂(¹⁵N), respectively, and the chemicalshift value of ¹H^(N) _(i) in a direct time domain dimension,t₃(¹H^(N)), and (2) to cosine modulate the chemical shift evolutions of¹³C^(α) _(i) and ¹³C^(β) _(l) in t₁(¹³C^(α/β)) with the chemical shiftevolutions of ¹H^(α) _(l) and ¹H^(β) _(i), respectively; and processingthe NMR signals to generate a 3D NMR spectrum with peak pairs derivedfrom said cosine modulating wherein (1) the chemical shift values of¹⁵N_(l) and ¹H^(N) _(l) are measured in two frequency domain dimensions,ω₂(¹⁵N) and ω₃(¹H^(N)), respectively, and (2) (i) the chemical shiftvalues of ¹H^(α) _(l) and ¹H^(β) _(i) are measured in a frequency domaindimension, ω₁(¹³C^(α/β)), by the frequency differences between the twopeaks forming said peak pairs, and (ii) the chemical shift values of¹³C^(α) _(l), and ¹³C^(β) _(i) are measured in a frequency domaindimension, ω₁(¹³C^(α/β)), by the frequencies at the center of said twopeaks forming said peak pairs.
 27. The method according to claim 26,wherein said applying radiofrequency pulses is carried out so that thechemical shift evolution of ¹⁵N_(l) does not occur and said processingthe NMR signals generates a two dimensional (2D) NMR spectrum with peakpairs wherein (1) the chemical shift value of ¹H^(N) _(i) is measured ina frequency domain dimension, ω₂(¹H^(N)), and (2) (i) the chemical shiftvalues of ¹H^(α) _(l) and ¹H^(β) _(i) are measured in a frequency domaindimension, ω₁(¹³C^(α/β)), by the frequency differences between the twopeaks forming said peak pairs, and (ii) the chemical shift values of¹³C^(α) _(i), and ¹³C^(β) _(i) are measured in a frequency domaindimension, ω₁(¹³C^(α/β)), by the frequencies at the center of the twopeaks forming said peak pairs.
 28. The method according to claim 26,wherein said applying radiofrequency pulses is carried out underconditions effective (1) to generate an additional NMR signal encodingthe chemical shift values of ¹³C^(α) _(i), ¹³C^(β) _(i) and ¹⁵N_(i) in aphase sensitive manner in t₁(¹³C^(α/β)) and t₂(¹⁵N) and the chemicalshift value of ¹H^(N) _(i) in t₃(¹H^(N)), and (2) to avoid cosinemodulating the chemical shift evolutions of ¹³C^(α) _(i) and ¹³C^(β)_(l) in t₁(¹³C^(α/β)) with the chemical shift evolutions of ¹H^(α) _(i)and ¹H^(β) _(i) for the additional NMR signal, and said processing theNMR signals and the additional NMR signal generates a 3D NMR spectrumwith additional peaks located centrally between the two peaks formingsaid peak pairs which measure the chemical shift values of ¹³C^(α) _(i)and ¹³C^(β) _(i) along ω₁(¹³C^(α/β)).
 29. The method according to claim28, wherein said additional peaks are derived from ¹³C^(α) and ¹³C^(β)nuclear spin polarization.
 30. The method according to claim 29, whereinsaid applying radiofrequency pulses effects a nuclear spin polarizationtransfer according to FIG. 1F, wherein a radiofrequency pulse is used tocreate transverse ¹H^(α) _(l) and ¹H^(β) _(l) magnetization, and ¹H^(α)_(l) and ¹H^(β) _(l) magnetization is transferred to ¹³C^(α) _(l) and¹³C^(β) _(l), to ¹⁵N_(i), and to ¹H^(N) _(l), where the NMR signal isdetected.
 31. The method according to claim 30, wherein said applyingradiofrequency pulses comprises: applying a first set of radiofrequencypulses according to the scheme shown in FIG. 2F to generate a first NMRsignal, and applying a second set of radiofrequency pulses according tothe scheme shown in FIG. 2F, wherein phase φ₁ of the first ¹H pulse isaltered by 180° to generate a second NMR signal, said method furthercomprising: adding and subtracting the first NMR signal and the secondNMR signal prior to said processing, whereby said processing the NMRsignals generates a first NMR subspectrum derived from said subtractingwhich contains said peak pairs, and a second NMR subspectrum derivedfrom said adding which contains said additional peaks located centrallybetween the two peaks forming said peak pairs.
 32. A method ofconducting a reduced dimensionality three-dimensional (3D) H,C,C,H-COSYnuclear magnetic resonance (NMR) experiment by measuring the chemicalshift values for ¹H^(m), ¹³C^(m), ¹H^(n), and ¹³C^(n) of a proteinmolecule wherein m and n indicate atom numbers of two CH, CH₂ or CH₃groups that are linked by a single covalent carbon—carbon bond in anamino acid residue, said method comprising: providing a protein sample;applying radiofrequency pulses to the protein sample which effect anuclear spin polarization transfer wherein the chemical shift evolutionsof ¹H^(m) and ¹³C^(m) are connected to the chemical shift evolutions of¹H^(n) and ¹³C^(n), under conditions effective (1) to generate NMRsignals encoding the chemical shift values of ¹³C^(m) and ¹³C^(n) in aphase sensitive manner in two indirect time domain dimensions,t₁(¹³C^(m)) and t₂(¹³C^(n)), respectively, and the chemical shift valueof ¹H^(n) in a direct time domain dimension, t₃(¹H^(n)), and (2) tocosine modulate the chemical shift evolution of ¹³C^(m) in t₁(¹³C^(m))with the chemical shift evolution of ¹H_(m); and processing the NMRsignals to generate a 3D NMR spectrum with peak pairs derived from saidcosine modulating wherein (1) the chemical shift values of ¹³C^(n) and¹H^(n) are measured in-two frequency domain dimensions, ω₂(¹³C^(n)) andω₃(¹H^(n)), respectively, and (2) the chemical shift values of ¹H^(m)and ¹³C^(m) are measured in a frequency domain dimension, ω₁(¹³C^(m)),by the frequency differences between the two peaks forming said peakpairs and the frequencies at the center of the two peaks, respectively.33. The method according to claim 32, wherein said applyingradiofrequency pulses is carried out so that the chemical shiftevolution of ¹³C^(n) does not occur and said processing the NMR signalsgenerates a two dimensional (2D) NMR spectrum with peak pairs wherein(1) the chemical shift value of ¹H^(n) is measured in a frequency domaindimension, ω₂(¹H^(n)), and (2) the chemical shift values of ¹H^(m) and¹³C^(m) are measured in a frequency domain dimension, ω₁(¹³C^(m)), bythe frequency differences between the two peaks forming said peak pairsand the frequencies at the center of the two peaks, respectively. 34.The method according to claim 32, wherein said applying radiofrequencypulses is carried out under conditions effective (1) to generate anadditional NMR signal encoding the chemical shift values of ¹³C^(m) and¹³C^(n) in a phase sensitive manner in t₁(¹³C^(m)) and t₂(¹³C^(n)) andthe chemical shift value of ¹H^(n) in t₃(¹H), and (2) to avoid cosinemodulating the chemical shift evolution of ¹³C^(m) in t₁(¹³C^(m)) withthe chemical shift evolution of ¹H^(m) for the additional NMR signal,and said processing the NMR signals and the additional NMR signalgenerates a 3D NMR spectrum with additional peaks located centrallybetween the two peaks forming said peak pairs which measure the chemicalshift value of ¹³C^(m) along ω₁(¹³C^(m)).
 35. The method according toclaim 34, wherein said additional peaks are derived from ¹³C^(m) nuclearspin polarization.
 36. The method according to claim 35, wherein saidapplying radiofrequency pulses effects a nuclear spin polarizationtransfer according to FIG. 1H, wherein a radiofrequency pulse is used tocreate transverse ¹H^(m) magnetization, and ¹H^(m) magnetization istransferred to ¹³C^(m), to ¹³C^(n), and to ¹H^(n), where the NMR signalis detected.
 37. The method according to claim 36, wherein said applyingradiofrequency pulses comprises: applying a first set of radiofrequencypulses according to the scheme shown in FIG. 2H to generate a first NMRsignal, and applying a second set of radiofrequency pulses according tothe scheme shown in FIG. 2H, wherein phase φ₁ of the first ¹H pulse isaltered by 180° to generate a second NMR signal, said method furthercomprising: adding and subtracting the first NMR signal and the secondNMR signal prior to said processing, whereby said processing the NMRsignals generates a first NMR subspectrum derived from said subtractingwhich contains said peak pairs, and a second NMR subspectrum derivedfrom said adding which contains said additional peaks located centrallybetween the two peaks forming said peak pairs.
 38. A method ofconducting a reduced dimensionality three-dimensional (3D) H,C,C,H-TOCSYnuclear magnetic resonance (NMR) experiment by measuring the chemicalshift values for ¹H^(m), ¹³C^(m), ¹H^(n), and ¹³C^(n), of a proteinmolecule wherein m and n indicate atom numbers of two CH, CH₂ or CH₃groups that may or may not be directly linked by a single covalentcarbon-carbon bond in an amino acid residue, said method comprising:providing a protein sample; applying radiofrequency pulses to theprotein sample which effect a nuclear spin polarization transfer whereinthe chemical shift evolutions of ¹H^(m) and ¹³C^(m) are connected to thechemical shift evolutions of ¹H^(n) and ¹³C^(n), under conditionseffective (1) to generate NMR signals encoding the chemical shift valuesof ¹³C^(m) and ¹³C^(n) in a phase sensitive manner in two indirect timedomain dimensions, t₁l(¹³C^(m)) and t₂(¹³C^(n)), and the chemical shiftvalue of ¹H^(n) in a direct time domain dimension, t₃(¹H^(n)), and (2)to cosine modulate the chemical shift evolution of ¹³C^(m) int₁(¹³C^(m)) with the chemical shift evolution of ¹H^(m); and processingthe NMR signals to generate a 3D NMR spectrum with peak pairs derivedfrom said cosine modulating wherein (1) the chemical shift values of¹³C^(n) and ¹H^(n) are measured in two frequency domain dimensions,ω₂(¹³C^(n)) and ω₃(¹H^(n)), respectively, and (2) the chemical shiftvalues of ¹H^(n) and ¹³C^(m) are measured in a frequency domaindimension, ω₁(¹³C^(m)), by the frequency differences between the twopeaks forming said peak pairs and the frequencies at the center of thetwo peaks, respectively.
 39. The method according to claim 38, whereinsaid applying radiofrequency pulses is carried out so that the chemicalshift evolution of ¹³C^(n) does not occur and said processing the NMRsignals generates a two dimensional (2D) NMR spectrum with peak pairswherein (1) the chemical shift value of ¹H^(n) is measured in afrequency domain dimension, ω₂(¹H^(n)), and (2) the chemical shiftvalues of ¹H^(m) and ¹³C^(m) are measured in a frequency domaindimension, ω₁(¹³C^(m)), by the frequency differences between the twopeaks forming said peak pairs and the frequencies at the center of thetwo peaks, respectively.
 40. The method according to claim 38, whereinsaid applying radiofrequency pulses is carried out under conditionseffective (1) to generate an additional NMR signal encoding the chemicalshift values of ¹³C^(m) and ¹³C^(n) in a phase sensitive manner int₁(¹³C^(m)) and t₂(¹³C^(n)) and the chemical shift value of ¹H^(n) int₃(¹H^(n)), and (2) to avoid cosine modulating the chemical shiftevolution of ¹³C^(m) in t₁(¹³C^(m)) with the chemical shift evolution of¹H^(m) for the additional NMR signal, and said processing the NMRsignals and the additional NMR signal generates a 3D NMR spectrum withadditional peaks located centrally between the two peaks forming saidpeak pairs which measure the chemical shift value of ¹³C^(m) alongω₁(¹³C^(m)).
 41. The method according to claim 40, wherein saidadditional peaks are derived from ¹³C^(m) nuclear spin polarization. 42.The method according to claim 41, wherein said applying radiofrequencypulses effects a nuclear spin polarization transfer according to FIG.1I, wherein a radiofrequency pulse is used to create transverse ¹H^(m)magnetization, and ¹H^(m) magnetization is transferred to ¹³C^(m), to¹³C^(n), and to ¹H^(n), where the NMR signal is detected.
 43. The methodaccording to claim 42, wherein said applying radiofrequency pulsescomprises: applying a first set of radiofrequency pulses according tothe scheme shown in FIG. 2I to generate a first NMR signal, and applyinga second set of radiofrequency pulses according to the scheme shown inFIG. 21, wherein phase φ₁ of the first ¹H pulse is altered by 180° togenerate a second NMR signal, said method further comprising: adding andsubtracting the first NMR signal and the second NMR signal prior to saidprocessing, whereby said processing the NMR signals generates a firstNMR subspectrum derived from said subtracting which contains said peakpairs, and a second NMR subspectrum derived from said adding whichcontains said additional peaks located centrally between the two peaksforming said peak pairs.
 44. A method of conducting a reduceddimensionality two-dimensional (2D) HB,CB,(CG,CD),HD nuclear magneticresonance (NMR) experiment by measuring the chemical shift values forthe following nuclei of a protein molecule : (1) a β-proton of an aminoacid residue with an aromatic side chain, ¹H^(β); (2) a β-carbon of anamino acid residue with an aromatic side chain, ¹³C^(β); and (3) aδ-proton of an amino acid residue with an aromatic side chain, ¹H^(δ),said method comprising: providing a protein sample; applyingradiofrequency pulses to the protein sample which effect a nuclear spinpolarization transfer wherein the chemical shift evolutions of ¹H^(β)and ¹³C^(β) are connected to the chemical shift evolution of ¹H^(δ),under conditions effective (1) to generate NMR signals encoding thechemical shift value of ¹³C^(β) in a phase sensitive manner in anindirect time domain dimension, t₁(¹³C^(β)), and the chemical shiftvalue of ¹H^(δ) in a direct time domain dimension, t₂(¹H^(δ)), and (2)to cosine modulate the chemical shift evolution of ¹³C^(β) int₁(¹³C^(β)) with the chemical shift evolution of ¹H^(β); and processingthe NMR signals to generate a 2D NMR spectrum with a peak pair derivedfrom said cosine modulating wherein (1) the chemical shift value of¹H^(δ) is measured in a frequency domain dimension, ω₂(¹H^(δ)), and (2)the chemical shift values of ¹H^(β) and ¹³C^(β) are measured in afrequency domain dimension, ω₁(¹³C^(β)), by the frequency differencebetween the two peaks forming said peak pair and the frequency at thecenter of the two peaks, respectively.
 45. The method according to claim44, wherein said applying radiofrequency pulses is carried out so that:(i) the chemical shift evolution of a δ-carbon of an amino acid residuewith an aromatic side chain, ¹³C^(δ), occurs under conditions effectiveto generate NMR signals encoding the chemical shift value of ¹³C^(δ) ina phase sensitive manner in an indirect time domain dimension,t₃(¹³C^(δ)), and said processing the NMR signals generates a threedimensional (3D) NMR spectrum with a peak pair wherein (1) the chemicalshift values of ¹H^(δ) and ¹³C^(δ) are measured in two frequency domaindimensions, ω₂(¹H^(δ)) and ω₃(¹³C^(δ)), respectively, and (2) thechemical shift values of ¹H^(β) and ¹³C^(β) are measured in a frequencydomain dimension, ω₁(¹³C^(β)), by the frequency difference between thetwo peaks forming said peak pair and the frequency at the center of thetwo peaks, respectively; or (ii) the chemical shift evolution of aγ-carbon of an amino acid residue with an aromatic side chain, ¹³C^(γ)occurs under conditions effective to generate NMR signals encoding thechemical shift value of ¹³C^(γ), in a phase sensitive manner in anindirect time domain dimension, t₃(¹³C^(γ)), and said processing the NMRsignals generates a three dimensional (3D) NMR spectrum with a peak pairwherein (1) the chemical shift values of ¹H^(δ) and ¹³C^(γ) are measuredin two frequency domain dimensions, ω₂(¹H^(δ)) and ω₃(¹³C^(γ)),respectively, and (2) the chemical shift values of ¹H^(β) and ¹³C^(β)are measured in a frequency domain dimension, ω₁(¹³C^(β)), by thefrequency difference between the two peaks forming said peak pair andthe frequency at the center of the two peaks, respectively.
 46. Themethod according to claim 44, wherein said applying radiofrequencypulses is carried out under conditions effective (1) to generate anadditional NMR signal encoding the chemical shift value of ¹³C^(β) in aphase sensitive manner in t₁(¹³C^(β)) and the chemical shift value of¹H^(δ) in t₂(¹H^(δ)), and (2) to avoid cosine modulating the chemicalshift evolution of ¹³C^(β) in t₁(¹³C^(β)) with the chemical shiftevolution of ¹H^(β) for the additional NMR signal, and said processingthe NMR signals and the additional NMR signal generates a 2D NMRspectrum with an additional peak located centrally between said peakpair which measure the chemical shift value of ¹³C^(β) alongω₁(¹³C^(β)).
 47. The method according to claim 46, wherein saidadditional peak is derived from ¹³C^(β) nuclear spin polarization. 48.The method according to claim 47, wherein said applying radiofrequencypulses effects a nuclear spin polarization transfer according to FIG.1J, wherein a radiofrequency pulse is used to create transverse ¹H^(β)magnetization, and ¹H^(β) magnetization is transferred to ¹³C^(β), to¹³C^(δ), and to ¹H^(δ), where the NMR signal is detected.
 49. The methodaccording to claim 48, wherein said applying radiofrequency pulsescomprises: applying a first set of radiofrequency pulses according tothe scheme shown in FIG. 2J to generate a first NMR signal, and applyinga second set of radiofrequency pulses according to the scheme shown inFIG. 2J, wherein phase φ₁ of the first ¹H pulse is altered by 180° togenerate a second NMR signal, said method further comprising: adding andsubtracting the first NMR signal and the second NMR signal prior to saidprocessing, whereby said processing the NMR signals generates a firstNMR subspectrum derived from said subtracting which contains said peakpair, and a second NMR subspectrum derived from said adding whichcontains said additional peak located centrally between the two peaksforming said peak pair.
 50. A method of conducting a reduceddimensionality two-dimensional (2D) H,C,H-COSY nuclear magneticresonance (NMR) experiment by measuring the chemical shift values for¹H^(m), ¹³C^(m), and ¹H^(n) of a protein molecule wherein m and nindicate atom numbers of two CH, CH₂ or CH₃ groups in an amino acidresidue, said method comprising: providing a protein sample; applyingradiofrequency pulses to the protein sample which effect a nuclear spinpolarization transfer wherein the chemical shift evolutions of ¹H^(m)and ¹³C^(m) are connected to the chemical shift evolution of ¹H^(n),under conditions effective (1) to generate NMR signals encoding thechemical shift value of ¹³C^(m) in a phase sensitive manner in anindirect time domain dimension, t₁(¹³C^(m)), and the chemical shiftvalue of ¹H^(n) in a direct time domain dimension, t₂(¹H^(n)), and (2)to cosine modulate the chemical shift evolution of ¹³C^(m) int₁(¹³C^(m)) with the chemical shift evolution of ¹H^(m); and processingthe NMR signals to generate a 2D NMR spectrum with peak pairs derivedfrom said cosine modulating wherein (1) the chemical shift value of¹H^(n) is measured in a frequency domain dimension, ω₂(¹H^(n)), and (2)the chemical shift values of ¹H^(m) and ¹³C^(m) are measured in afrequency domain dimension, ω₁(¹³C^(m)), by the frequency differencesbetween the two peaks forming said peak pairs and the frequencies at thecenter of the two peaks, respectively.
 51. The method according to claim50, wherein said applying radiofrequency pulses effects a nuclear spinpolarization transfer according to FIG. 1K, wherein a radiofrequencypulse is used to create transverse ¹H^(m) magnetization, and ¹H^(m)polarization is transferred to ¹³C^(m), to ¹H^(m), and to ¹H^(n), wherethe NMR signal is detected.
 52. The method according to claim 51,wherein said applying radiofrequency pulses is carried out according tothe scheme shown in FIG. 2K.
 53. A method for sequentially assigningchemical shift values of an α-proton, ¹H^(α), an α-carbon, ¹³C^(α), apolypeptide backbone amide nitrogen, ¹⁵N, and a polypeptide backboneamide proton, ¹H^(N), of a protein molecule comprising: providing aprotein sample; conducting a set of reduced dimensionality (RD) nuclearmagnetic resonance (NMR) experiments on the protein sample comprising:(1) a RD three dimensional (3D) HA,CA,(CO),N,HN NMR experiment tomeasure and connect chemical shift values of the α-proton of amino acidresidue i−1, ¹H^(α) _(l−1), the α-carbon of amino acid residue i−1,¹³C^(α) _(i−1), the polypeptide backbone amide nitrogen of amino acidresidue i, ¹⁵N_(i), and the polypeptide backbone amide proton of aminoacid residue i, ¹H^(N) _(i) and (2) a RD 3D HNNCAHA NMR experiment tomeasure and connect the chemical shift values of the α-proton of aminoacid residue i, ¹H^(α) _(i), the α-carbon of amino acid residue i,¹³C^(α) _(i), ¹⁵N_(i), and ¹H^(N) _(i); and obtaining sequentialassignments of the chemical shift values of ¹H^(α), ¹³C^(α), ¹⁵N, and¹H^(N) by (i) matching the chemical shift values of ¹H^(α) _(l−1) and¹³C^(α) _(i−1) with the chemical shift values of ¹H^(α) _(i) and ¹³C^(α)_(i), (ii) using the chemical shift values of ¹H^(α) _(i−1) and ¹³C^(α)_(i−1) to identify the type of amino acid residue i−1, and (iii) mappingsets of sequentially connected chemical shift values to the amino acidsequence of the polypeptide chain and using said chemical shift valuesto locate secondary structure elements within the polypeptide chain. 54.The method according to claim 53 further comprising: subjecting theprotein sample to a RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment tomeasure and connect the chemical shift values of the β-proton of aminoacid residue i−1, ¹H^(β) _(i−1), the β-carbon of amino acid residue i−1,¹³C^(β) _(i−1), ¹H^(α) _(i−1), ¹³C^(α) _(i−1), ¹⁵N_(i), and ¹H^(N) _(i);and obtaining sequential assignments of the chemical shift values of¹H^(β) and ¹³C^(β) by using the chemical shift values of ¹H^(β) _(l−1)and ¹³C^(β) _(l−1) to identify the type of amino acid residue i−1. 55.The method according to claim 54 further comprising: subjecting theprotein sample to a RD 3D H ^(α/β),C ^(α/β),CO,HA NMR experiment tomeasure and connect the chemical shift values of the β-proton of aminoacid residue i, ¹H^(β) _(l), the β-carbon of amino acid residue i,¹³C^(β) _(l), ¹H^(α) _(i), ¹³C^(α) _(i), and a polypeptide backbonecarbonyl carbon of amino acid residue i, ¹³C′_(l); and obtainingsequential assignments of the chemical shift value of ¹³C′_(i) bymatching the chemical shift values of ¹H^(β) _(i), ¹³C^(β) _(i), ¹H^(α)_(i), and ¹³C^(α) _(i) measured by said RD 3D H ^(α/β),C ^(α/β),CO,HANMR experiment with the sequentially assigned chemical shift values of¹H^(β), ¹³C^(β), ¹H^(α), ¹³C^(α), ¹⁵N, and ¹H^(N) measured by said RD 3DHA,CA,(CO),N,HN NMR experiment, RD 3D HNNCAHA NMR experiment, and RD 3DH ^(α/β) C ^(α/β)(CO)NHN NMR experiment.
 56. The method according toclaim 54 further comprising: subjecting the protein sample to a RD 3D H^(α/β), C ^(α/β),N,HN NMR experiment to measure and connect the chemicalshift values of ¹H^(β) _(l), ¹³C^(β) _(l), ¹H^(α) _(i), ¹³C^(α) _(l),¹⁵N_(l), and ¹H^(N) _(i); and obtaining sequential assignments bymatching the chemical shift values of ¹H^(β) _(i), ¹³C^(β) _(i), ¹H^(α)_(i), and ¹³C^(α) _(i) with the chemical shift values of ¹H^(β) _(i−1),¹³C^(β) _(l−1), ¹H^(α) _(i−1), and ¹³C^(α) _(i−1) measured by said RD 3DH ^(α/β) C ^(α/β)(CO)NHN NMR experiment.
 57. The method according toclaim 54 further comprising: subjecting the protein sample to a 3DHNNCACB NMR experiment to measure and connect the chemical shift valueof ¹³C^(β) _(l), ¹³C^(α) _(i), ¹⁵N_(i), and ¹H^(N) _(l); and obtainingsequential assignments by matching the chemical shift values of ¹³C^(β)_(i) and ¹³C^(α) _(i) measure by said 3D HNNCACB NMR experiment with thechemical shift values of ¹³C^(β) _(i−1) and ¹³C^(α) _(i−1) measured bysaid RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment.
 58. The methodaccording to claim 54 further comprising: subjecting the protein sampleto a RD two-dimensional (2D) HB,CB,(CG,CD),HD NMR experiment to measureand connect the chemical shift values of ¹H^(β) _(l−1), ¹³C^(β) _(l−1),and a δ-proton of amino acid residue i−1 with an aromatic side chain,¹H^(δ) _(l−1); and obtaining sequential assignments by (i) matching thechemical shift values of ¹H^(β) _(i−1) and ¹³C^(β) _(i−1) measured bysaid RD 2D HB, CB, (CG,CD), HD NMR experiment with the chemical shiftvalues of ¹H^(β) and ¹³C^(β) measured by said RD 3D H ^(α/β) C^(α/β)(CO)NHN NMR experiment, (ii) using said chemical shift values toidentify amino acid residue i as having an aromatic side chain, and(iii) mapping sets of sequentially connected chemical shift values tothe amino acid sequence of the polypeptide chain and locating amino acidresidues with aromatic side chains along said polypeptide chain.
 59. Themethod according to claim 54 further comprising: subjecting the proteinsample to a RD 3D H,C,C,H-COSY NMR experiment or a RD 3D H,C,C,H-TOCSYNMR experiment to measure and connect the chemical shift values ofaliphatic protons of amino acid residue i, ¹H^(ali) _(i), and aliphaticcarbons of amino acid residue i, ¹³C^(ali) _(l); and obtainingsequential assignments of the chemical shift values of ¹H^(ali) _(i) and¹³C^(ali) _(i), by (i) matching the chemical shift values of ¹H^(β)_(i), ¹³C^(β) _(l), ¹H^(α) _(i), and ¹³C^(α) _(i) measured using said RD3D H,C,C,H-COSY NMR experiment or RD 3D H,C,C,H-TOCSY RD NMR experimentwith the chemical shift values of ¹H^(β), ¹³C^(β), ¹H^(α), and ¹³C^(α)measured by said RD 3D HA,CA,(CO),N,HN NMR experiment, RD 3D HNNCAHA NMRexperiment, and RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment and (ii)using the chemical shift values of ¹H^(ali) _(i) and ¹³C^(ali) _(i), toidentify the type of amino acid residue i.
 60. The method according toclaim 53 further comprising: subjecting the protein sample to a RD 3DHNN<CO,CA> NMR experiment to measure and connect the chemical shiftvalues of a polypeptide backbone carbonyl carbon of amino acid residuei−1, ¹³C′_(i−)1, ¹³C^(α) _(l), ¹⁵N_(l), and ¹H^(N) _(i); and obtainingsequential assignments of the chemical shift value of ¹³C′_(i−1) bymatching the chemical shift value of ¹³C^(α) _(l) measured by said RD 3DHNN<CO,CA> NMR experiment with the sequentially assigned chemical shiftvalues of ¹³C^(α), ¹⁵N, and ¹H^(N) measured by said RD 3DHA,CA,(CO),N,HN NMR experiment and RD 3D HNNCAHA NMR experiment.
 61. Themethod according to claim 53 further comprising: subjecting the proteinsample to (i) a RD 3D H ^(α/β),C ^(α/β),CO,HA NMR experiment to measureand connect the chemical shift values of the β-proton of amino acidresidue i, ¹H^(β) _(l), the β-carbon of amino acid residue i, ¹³C^(β)_(l), the α-proton of amino acid residue i, ¹H^(α) _(l), the α-carbon ofamino acid residue i, ¹³C^(α) _(l), and a polypeptide backbone carbonylcarbon of amino acid residue i, ¹³C′_(i) and (ii) a RD 3D HNN<CO,CA> NMRexperiment to measure and connect the chemical shift values of ¹³C′_(l),the α-carbon of amino acid residue i+1, ¹³C^(α) _(i+1), the polypeptidebackbone amide nitrogen of amino acid residue i+1, ¹⁵N_(i+1), and thepolypeptide backbone amide proton of amino acid residue i+1, ¹H^(N)_(i+1); and obtaining sequential assignments by matching the chemicalshift value of ¹³C′_(i) measured by said RD 3D HNN<CO,CA> NMR experimentwith the chemical shift value of ¹³C′_(i) measured by said RD 3D H^(α/β),C ^(α/β),CO,HA NMR experiment.
 62. The method according to claim53, further comprising: subjecting the protein sample to a RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment to measure and connect the chemicalshift values of aliphatic protons of amino acid residue i−1, ¹H^(ali)_(l−1), aliphatic carbons of amino acid residue i−1, ¹³C^(ali) _(i−1),¹⁵N_(l), and ¹H^(N) _(i); and obtaining sequential assignments of thechemical shift values of ¹H^(ali) _(l−1) and ¹³C^(ali) _(i−1) for aminoacid residues i having unique pairs of ¹⁵N_(i) and ¹H^(N) _(i) chemicalshift values by matching the chemical shift values of ¹H^(α) and ¹³C^(α)measured by said RD 3D HNNCAHA NMR experiment and RD 3D HA,CA,(CO),N,HNNMR experiment with the chemical shift values of ¹H^(α) _(i−1) and¹³C^(α) _(l−1) measured by said RD 3D H,C,(C-TOCSY-CO),N,HN NMRexperiment and using the ¹H^(ali) _(i−1) and ¹³C^(ali) _(i−1) chemicalshift values to identify the type of amino acid residue i−1.
 63. Themethod according to claim 53 further comprising: subjecting the proteinsample to a RD 3D H,C,C,H-COSY NMR experiment or a RD 3D H,C,C,H-TOCSYNMR experiment to measure and connect the chemical shift values ofaliphatic protons of amino acid residue i, ¹H^(ali) _(i), and aliphaticcarbons of amino acid residue i, ¹³C^(ali) _(l); and obtainingsequential assignments of the chemical shift values of ¹H^(ali) _(i) and¹³C^(ali) _(l) by (i) matching the chemical shift values of ¹H^(α) _(l)and ¹³C^(α) _(l) measured using said RD 3D H,C,C,H-COSY NMR experimentor RD 3D H,C,C,H-TOCSY RD NMR experiment with the chemical shift valuesof ¹H^(α) and ¹³C^(α) measured by said RD 3D HA,CA,(CO),N,HN NMRexperiment and RD 3D HNNCAHA NMR experiment and (ii) using the chemicalshift values of ¹H^(ali) _(i) and ¹³C^(ali) _(i), to identify the typeof amino acid residue i.
 64. A method for sequentially assigningchemical shift values of a β-proton, ¹H^(β), a β-carbon, ¹³C^(β), anα-proton, ¹H^(α), an α-carbon, ¹³C^(α), a polypeptide backbone amidenitrogen, ¹⁵N, and a polypeptide backbone amide proton, ¹H^(N), of aprotein molecule comprising: providing a protein sample; conducting aset of reduced dimensionality (RD) nuclear magnetic resonance (NMR)experiments on the protein sample comprising: (1) a RD three-dimensional(3D) H ^(α/β) C ^(α/β)(CO)NHN NMR experiment to measure and connect thechemical shift values of the β-proton of amino acid residue i−1, ¹H^(β)_(i−1), the β-carbon of amino acid residue i−1, ¹³C^(β) _(l−1), theα-proton of amino acid residue i−1, ¹H^(α) _(l−1), the α-carbon of aminoacid residue i−1, ¹³C^(α) _(i−1), the polypeptide backbone amidenitrogen of amino acid residue i, ¹⁵N_(i), and the polypeptide backboneamide proton of amino acid residue i, ¹H^(N) _(i) and (2) a RD 3D H^(α/β),C ^(α/β),N,HN NMR experiment to measure and connect the chemicalshift values of the β-proton of amino acid residue i, ¹H^(β) _(i), theβ-carbon of amino acid residue i, ¹³C^(β) _(i), the α-proton of aminoacid residue i, ¹H^(α) _(i), the α-carbon of amino acid residue i,¹³C^(α) _(i), ¹⁵N_(l), and ¹H^(N) _(l); and obtaining sequentialassignments of the chemical shift values of ¹H^(β), ¹³C^(β), ¹H^(α),¹³C^(α), ¹⁵N, and ¹H^(N) by (i) matching the chemical shift values ofthe α- and β-protons of amino acid residue i−1, ¹H^(α/β) _(i−1), and theα- and β-carbons of amino acid residue i−1, ¹³C^(α/β) _(i−1), with thechemical shift values of ¹H^(α/β) _(i) and ¹³C^(α/β) _(i), (ii) usingthe chemical shift values of ¹H^(α/β) _(i−1) and ¹³C^(α/β) _(i−1) toidentify the type of amino acid residue i−1, and (iii) mapping sets ofsequentially connected chemical shift values to the amino acid sequenceof the polypeptide chain and using said chemical shift values to locatesecondary structure elements within the polypeptide chain.
 65. Themethod according to claim 64 further comprising: subjecting the proteinsample to a RD 3D HA,CA,(CO),N,HN NMR experiment (i) to measure andconnect chemical shift values of ¹H^(α) _(l−1), ¹³C^(α) _(l−1), ¹⁵N_(l),and ¹H^(N) _(l) and (ii) to distinguish between NMR signals for¹H^(α)/¹³C^(α) and ¹H^(β)/¹³C^(β) measured in said RD 3D H ^(α/β) C^(α/β)(CO)NHN NMR experiment and RD 3D H ^(α/β),C ^(α/β),N,HN NMRexperiment.
 66. The method according to claim 64 further comprising:subjecting the protein sample to a RD 3D H ^(α/β),C ^(α/β),CO,HA NMRexperiment to measure and connect the chemical shift values of ¹H^(β)_(i), ¹³C^(β) _(i), ¹H^(α) _(l), ¹³C^(α) _(i), and a polypeptidebackbone carbonyl carbon of amino acid residue i, ¹³C′_(l); andobtaining sequential assignments of the chemical shift value of ¹³C′_(l)by matching the chemical shift values of ¹H^(β) _(l), ¹³C^(β) _(i),¹H^(α) _(i), and ¹³C^(α) _(i) measured by said RD 3D H ^(α/β),C^(α/β),C^(α/β),CO,HA NMR experiment with the sequentially assignedchemical shift values of ¹H^(β), ¹³C^(β), ¹H^(α), ¹³C^(α), ¹⁵N, and¹H^(N) measured by said RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experimentand RD 3D H ^(α/β),C ^(α/β),N,HN NMR experiment.
 67. The methodaccording to claim 64 further comprising: subjecting the protein sampleto a RD 3D NN <CO,CA> NMR experiment to measure and connect the chemicalshift values of a polypeptide backbone carbonyl carbon of amino acidresidue i−1, ¹³C′_(l−1), ¹³C^(α) _(i), ¹⁵N_(l), and ¹H^(N) _(i); andobtaining sequential assignments of the chemical shift value of¹³C′_(l−1) by matching the chemical shift value of ¹³C^(α) _(i) measuredby said RD 3D HNN<CO,CA> NMR experiment with the sequentially assignedchemical shift values of ¹³C^(α), ¹⁵N, and ¹H^(N) measured by said RD 3DH ^(α/β) C ^(α/β)(CO)NHN NMR experiment and RD 3D H ^(α/β),C ^(α/β),N,HNNMR experiment.
 68. The method according to claim 64 further comprising:subjecting the protein sample to (i) a RD 3D H^(α/β),C^(α/β),CO,HA NMRexperiment to measure and connect the chemical shift values of ¹H^(β)_(l), ¹³C^(β) _(i), ¹H^(α) _(i), ¹³C^(α) _(l), and a polypeptidebackbone carbonyl carbon of amino acid residue i, ¹³C′_(i) and (ii) a RD3D HNN<CO,CA> NMR experiment to measure and connect the chemical shiftvalues of ¹³C′_(i), the α-carbon of amino acid residue i+1, ¹³C^(α)_(i+1), the polypeptide backbone amide nitrogen of amino acid residuei+l, ¹⁵N_(l+1), and the polypeptide backbone amide proton of amino acidresidue i+1, ¹H^(N) _(l+1); and obtaining sequential assignments bymatching the chemical shift value of ¹³C′_(i) measured by said RD 3DHNN<CO,CA> NMR experiment with the chemical shift value of ¹³C′_(l)measured by said RD 3D H ^(α/β),C ^(α/β),CO,HA NMR experiment.
 69. Themethod according to claim 64 further comprising: subjecting the proteinsample to a RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment to measure andconnect the chemical shift values of ¹H^(ali) _(l−1), ¹³C^(ali) _(i−1),¹⁵N_(l), and ¹H^(N) _(l); and obtaining sequential assignments of thechemical shift values of ¹H^(ali) _(i−1) and ¹³C^(ali) _(i−1) for aminoacid residues i having unique pairs of ¹⁵N_(i) and ¹H^(N) _(i) chemicalshift values by matching the chemical shift values of ¹H^(β), ¹³C^(β),¹H^(α), and ¹³C^(α) measured by said RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMRexperiment and RD 3D H ^(α/β),C ^(α/β),N,HN NMR experiment with thechemical shift values of ¹H^(β) _(i−1), ¹³C^(β) _(i−1), ¹H^(α) _(i−1),and ¹³C^(α) _(i−1), measured by said RD 3D H,C,(C-TOCSY-CO),N,HN NMRexperiment and using the ¹H^(ali) _(i−1), and ¹³C^(ali) _(i−1) chemicalshift values to identify the type of amino acid residue i−1.
 70. Themethod according to claim 64 further comprising: subjecting the proteinsample to a 3D HNNCACB NMR experiment to measure and connect thechemical shift value of ¹³C^(β) _(l), ¹³C^(α) _(l), ¹⁵N_(l), and ¹H^(N)_(l); and obtaining sequential assignments by matching the chemicalshift values of ¹³C^(β) _(i) and ¹³C^(α) _(i) measured by said 3DHNNCACB NMR experiment with the chemical shift values of ¹³C^(β) _(i−1)and ¹³C^(α) _(i−1) measured by said RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMRexperiment.
 71. The method according to claim 64 further comprising:subjecting the protein sample to a RD two-dimensional (2D)HB,CB,(CG,CD),HD NMR experiment to measure and connect the chemicalshift values of ¹H^(β) _(l), ¹³C^(β) _(l), and a δ-proton of amino acidresidue i with an aromatic side chain, ¹H^(δ) _(i); and obtainingsequential assignments by (i) matching the chemical shift values of¹H^(β) _(l) and ¹³C^(β) _(l) measured by said RD 2D HC, CB, (CG, CD),HDNMR experiment with the chemical shift values of ¹H^(β) and ¹³C^(β)measured by said RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment and RD 3DH ^(α/β),C ^(α/β),N,HN NMR experiment, (ii) using said chemical shiftvalues to identify amino acid residue i as having an aromatic sidechain, and (iii) mapping sets of sequentially connected chemical shiftvalues to the amino acid sequence of the polypeptide chain and locatingamino acid residues with aromatic side chains along said polypeptidechain.
 72. The method according to claim 64, further comprising:subjecting the protein sample to a RD 3D H,C,C,H-COSY NMR experiment ora RD 3D H,C,C,H-TOCSY NMR experiment to measure and connect the chemicalshift values of aliphatic protons of amino acid residue i, ¹H^(ali) _(i)and aliphatic carbons of amino acid residue i, ¹³C^(ali) _(i); andobtaining sequential assignments of the chemical shift values of¹H^(ali) _(i) and ¹³C^(ali) _(l) by (i) matching the chemical shiftvalues of ¹H^(β) _(i), ¹³C^(β) _(i), ¹H^(α) _(i), and ¹³C^(α) _(l)measured using said RD 3D H,C,C,H-COSY NMR experiment or RD 3DH,C,C,H-TOCSY RD NMR experiment with the chemical shift values of¹H^(β), ¹³C^(β), ¹H^(α), and ¹³C^(α) measured by said RD 3D H ^(α/β) C^(α/β)(CO)NHN NMR experiment and RD 3D H ^(α/β),C ^(α/β),N,HN NMRexperiment, and (ii) using the chemical shift values of ¹H^(ali) _(i)and ¹³C^(ali) _(i), to identify the type of amino acid residue i.
 73. Amethod for sequentially assigning the chemical shift values of aliphaticprotons, ¹H^(ali), aliphatic carbons, ¹³C^(ali), a polypeptide backboneamide nitrogen, ¹⁵N, and a polypeptide backbone amide proton, ¹H^(N), ofa protein molecule comprising: providing a protein sample; conducting aset of reduced dimensionality (RD) nuclear magnetic resonance (NMR)experiments on the protein sample comprising: (1) a RD three-dimensional(3D) H,C,(C-TOCSY-CO),N,HN NMR experiment to measure and connect thechemical shift values of the aliphatic protons of amino acid residuei−1, ¹H^(ali) _(l−1), the aliphatic carbons of amino acid residue i−1,¹³C^(ali) _(l−1), the polypeptide backbone amide nitrogen of amino acidresidue i, ¹⁵N_(l), and the polypeptide backbone amide proton of aminoacid residue i, ¹H^(N) _(i) and (2) a RD 3D H ^(α/β),C ^(α/β),N,HN NMRexperiment to measure and connect the chemical shift values of theβ-proton of amino acid residue i, ¹H^(β) _(i), the β-carbon of aminoacid residue i, ¹³C^(β) _(i), the α-proton of amino acid residue i,¹H^(α) _(i), the α-carbon of amino acid residue i, ¹³C^(α) _(i),¹⁵N_(l), and ¹H^(N) _(l); and obtaining sequential assignments of thechemical shift values of ¹H^(ali), ¹³C^(ali), ⁵N, and ¹H^(N) by (i)matching the chemical shift values of the α- and β-protons of amino acidresidue i−1, ¹H^(α/β) _(l−1) and the α- and β-carbons of amino acidresidue i−1, ¹³C^(α/β) _(i−1) with the chemical shift values of ¹H^(α/β)_(i) and ¹³C^(α/β) _(i) of amino acid residue i, (ii) using the chemicalshift values of ¹H^(ali) _(i−1) and ¹³C^(ali) _(l−1) to identify thetype of amino acid residue i−1, and (iii) mapping sets of sequentiallyconnected chemical shift values to the amino acid sequence of thepolypeptide chain and using said chemical shift values to locatesecondary structure elements within the polypeptide chain.
 74. Themethod according to claim 73 further comprising: subjecting the proteinsample to a RD 3D H ^(α/β),C ^(α/β),CO,HA NMR experiment to measure andconnect the chemical shift values of ¹H^(β) _(i), ¹³C^(β) _(l), ¹H^(α)_(l), ¹³C^(α) _(i), and a polypeptide backbone carbonyl carbon of aminoacid residue i, ¹³C′_(i); and obtaining sequential assignments of thechemical shift value of ¹³C′_(l) by matching the chemical shift valuesof ¹H^(β) _(i), ¹³C^(β) _(i), ¹H^(α) _(i), and ¹³C^(α) _(i) measured bysaid RD 3D H ^(α/β),C ^(α/β),CO,HA NMR experiment with the sequentiallyassigned chemical shift values of ¹H^(β), ¹³C^(β), ¹H^(α), ¹³C^(α), ¹⁵N,and ¹H^(N) measured by said RD 3D H,C,(C-TOCSY-CO),N,HN NMR experimentand RD 3D H ^(α/β),C ^(α/β),N,HN NMR experiment.
 75. The methodaccording to claim 73 further comprising: subjecting the protein sampleto a RD 3D HNN<CO,CA> NMR experiment to measure and connect the chemicalshift values of a polypeptide backbone carbonyl carbon of amino acidresidue i−1, ¹³C′_(l−1), ¹³C^(α) _(i), ¹⁵N_(i), and ¹H^(N) _(l); andobtaining sequential assignments of the chemical shift value of¹³C′_(i−1) by matching the chemical shift value of ¹³C^(α) _(l) measuredby said RD 3D HNN<CO,CA> NMR experiment with the sequentially assignedchemical shift values of ¹³C^(α), ¹⁵N, and ¹H^(N) measured by said RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment and RD 3D H ^(α/β),C ^(α/β),N,HNNMR experiment.
 76. The method according to claim 73 further comprising:subjecting the protein sample to (i) a RD 3D H ^(α/β),C ^(α/β),CO,HA NMRexperiment to measure and connect the chemical shift values of ¹H^(β)_(i), ¹³C^(β) _(l), ¹H^(α) _(l), ¹³C^(α) _(l), and a polypeptidebackbone carbonyl carbon of amino acid residue i, ¹³C′_(i) and (ii) a RD3D HNN<CO,CA> NMR experiment to measure and connect the chemical shiftvalues of ¹³C′_(l), the α-carbon of amino acid residue i+1, ¹³C^(α)_(l+1), the polypeptide backbone amide nitrogen of amino acid residuei+1, ¹⁵N_(i+1), and the polypeptide backbone amide proton of amino acidresidue i+1, ¹H^(N) _(l+1); and obtaining sequential assignments bymatching the chemical shift value of ¹³C′_(i) measured by said RD 3DHNN<CO,CA> NMR experiment with the chemical shift value of ¹³C′_(l)measured by said RD 3D H ^(α/β),C ^(α/β),CO,HA NMR experiment.
 77. Themethod according to claim 73 further comprising: subjecting the proteinsample to a RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment (i) to measureand connect the chemical shift values of ¹ H^(α/β) _(i−1), ¹³C^(α/β)_(i−1), ¹⁵N_(i), and ¹H^(N) _(l), and (ii) to identify NMR signals for¹H^(α/β) _(i−1), ¹³C^(α/β) _(l−1), ¹⁵N_(i), and ¹H^(N) _(i) in said RD3D H,C,(C-TOCSY-CO),N,HN NMR experiment.
 78. The method according toclaim 73 further comprising: subjecting the protein sample to a RD 3DHA,CA,(CO),N,HN NMR experiment (i) to measure and connect chemical shiftvalues of ¹H^(α) _(l−1), ¹³C^(α) _(i−1), ¹⁵N_(i), and ¹H^(N) _(i) and(ii) to identify NMR signals for ¹H^(α) and ¹³C^(α) in said RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment and RD 3D H ^(α/β),C ^(α/β),N,HNNMR experiment.
 79. The method according to claim 73 further comprising:subjecting the protein sample to a 3D HNNCACB NMR experiment to measureand connect the chemical shift value of ¹³C^(β) _(i), ¹³C^(α) _(i),¹⁵N_(i), and ¹H^(N) _(l); and obtaining sequential assignments bymatching the chemical shift values of ¹³C^(β) _(i) and ¹³C^(α) _(i)measured by said 3D HNNCACB NMR experiment with the chemical shiftvalues of ¹³C^(β) _(i−1), and ¹³C^(α) _(i−1) measured by said RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment.
 80. The method according to claim73 further comprising: subjecting the protein sample to a RDtwo-dimensional (2D) HB,CB,(CG,CD),HD NMR experiment to measure andconnect the chemical shift values of ¹H^(β) _(i), ¹³C^(β) _(l), and aδ-proton of amino acid residue i with an aromatic side chain, ¹H^(δ)_(i); and obtaining sequential assignments by matching the chemicalshift values of ¹H^(β) _(i) and ¹³C^(β) _(l) measured by said RD 2D HB,CB, (CG, CD), HD NMR experiment with the chemical shift values of ¹H^(β)and ¹³C^(β) measured by said RD 3D H ^(α/β), C ^(α/β),N,HN NMRexperiment and RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, using saidchemical shift values to identify amino acid residue i as having anaromatic side chain, and mapping sets of sequentially connected chemicalshift values to the amino acid sequence of the polypeptide chain andlocating amino acid residues with aromatic side chains along saidpolypeptide chain.
 81. The method according to claim 73 furthercomprising: subjecting the protein sample to a RD 3D H,C,C,H-COSY NMRexperiment or a RD 3D H,C,C,H-TOCSY NMR experiment to measure andconnect the chemical shift values of aliphatic protons of amino acidresidue i, ¹H^(ali) _(i) and aliphatic carbons of amino acid residue i,¹³C^(ali) _(i); and obtaining sequential assignments of the chemicalshift values of ¹H^(ali) _(l) and ¹³C^(ali) _(i) by (i) matching thechemical shift values of ¹H^(ali) _(i) and ¹³C^(ali) _(l) measured usingsaid RD 3D H,C,C,H-COSY NMR experiment or RD 3D H,C,C,H-TOCSY NMRexperiment with the chemical shift values of ¹H^(ali) and ¹³C^(ali)measured by said RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and RD 3D H^(α/β),C ^(α/β),N,HN NMR experiment, and (ii) using the chemical shiftvalues of ¹H^(ali) _(i) and ¹³C^(ali) _(i), to identify the type ofamino acid residue i.
 82. A method for sequentially assigning chemicalshift values of aliphatic protons, ¹H^(ali), aliphatic carbons,¹³C^(ali), a polypeptide backbone amide nitrogen, ¹⁵N, and a polypeptidebackbone amide proton, ¹H^(N), of a protein molecule comprising:providing a protein sample; conducting a set of reduced dimensionality(RD) nuclear magnetic resonance (NMR) experiments on the protein samplecomprising: (1) a RD three-dimensional (3D) H,C,(C-TOCSY-CO),N,HN NMRexperiment to measure and connect the chemical shift values of thealiphatic protons of amino acid residue i−1, ¹H^(ali) _(l−1), thealiphatic carbons of amino acid residue i−1, ¹³C^(ali) _(l−1), thepolypeptide backbone amide nitrogen of amino acid residue i, ¹⁵N_(i),and the polypeptide backbone amide proton of amino acid residue i,¹H^(N) _(i) and (2) a RD 3D HNNCAHA NMR experiment to measure andconnect the chemical shift values of the α-proton of amino acid residuei, ¹H^(α) _(l), the α-carbon of amino acid residue i, ¹³C^(α) _(i),¹⁵N_(l), and ¹H^(N) _(l); and obtaining sequential assignments of thechemical shift values of ¹H^(ali), ¹³C^(ali), ¹⁵N, and ¹H^(N) by (i)matching the chemical shift values of the a-proton of amino acid residuei−1, ¹H^(α) _(i−1), and the α-carbon of amino acid residue i−1, ¹³C^(α)_(i−1), with the chemical shift values of ¹H^(α) _(i) and ¹³C^(α) _(l),(ii) using the chemical shift values of ¹H^(ali) _(l−1), and ¹³C^(ali)_(i−1) to identify the type of amino acid residue i−1, and (iii) mappingsets of sequentially connected chemical shift values to the amino acidsequence of the polypeptide chain and using said chemical shift valuesto locate secondary structure elements within the polypeptide chain. 83.The method according to claim 82 further comprising: subjecting theprotein sample to a RD 3D H ^(α/β),C ^(α/β),CO,HA NMR experiment tomeasure and connect the chemical shift values of a β-proton of aminoacid residue i, ¹H^(β) _(i), a β-carbon of amino acid residue i, ¹³C^(β)_(l), ¹H^(α) _(i), ¹³C^(α) _(i), and a polypeptide backbone carbonylcarbon of amino acid residue i, ¹³C′_(i); and obtaining sequentialassignments of the chemical shift value of ¹³C′_(l) by matching thechemical shift values of ¹H^(β) _(l), ¹³C^(β) _(i), ¹H^(α) _(i), and¹³C^(α) _(i) measured by said RD 3D H ^(α/β),C ^(α/β),CO,HA NMRexperiment with the sequentially assigned chemical shift values of¹H^(β), ¹³C^(β), ¹H^(α), ¹³C^(α), ¹⁵N, and ¹H^(N) measured by said RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment and RD 3D HNNCAHA NMR experiment.84. The method according to claim 82 further comprising: subjecting theprotein sample to a RD 3D HNN<CO,CA> NMR experiment to measure andconnect the chemical shift values of a polypeptide backbone carbonylcarbon of amino acid residue i−1, ¹³C′_(l−1), ¹³C^(α) _(l), ¹⁵N_(l), and¹H^(N) _(i); and obtaining sequential assignments of the chemical shiftvalue of ¹³C′_(l−1) by matching the chemical shift value of ¹³C^(α) _(i)measured by said RD 3D HNN<CO,CA> NMR experiment with the sequentiallyassigned chemical shift values of ¹³C^(α), ¹⁵N, and ¹H^(N) measured bysaid RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and RD 3D HNNCAHA NMRexperiment.
 85. The method according to claim 82 further comprising:subjecting the protein sample to (i) a RD 3D H ^(α/β),C ^(α/β),CO,HA NMRexperiment to measure and connect the chemical shift values of aβ-proton of amino acid residue i, ¹H^(β) _(l), β-carbon of amino acidresidue i, ¹³C^(β) _(i), the α-proton of amino acid residue i, ¹H^(α)_(i), the α-carbon of amino acid residue i, ¹³C^(α) _(i), and apolypeptide backbone carbonyl carbon of amino acid residue i, ¹³C′_(i)and (ii) a RD 3D HNN<CO,CA> NMR experiment to measure and connect thechemical shift values of ¹³C′_(l), an α-carbon of amino acid residuei+1, ¹³C^(α) _(i+1), a polypeptide backbone amide nitrogen of amino acidresidue i+1, ¹⁵N_(l+1), and a polypeptide backbone amide proton of aminoacid residue i+1, ¹H^(N) _(l+1); and obtaining sequential assignments bymatching the chemical shift value of ¹³C′_(l) measured by said RD 3DHNN<CO,CA> NMR experiment with the chemical shift value of ¹³C′_(l)measured by said RD 3D H ^(α/β),C ^(α/β),CO,HA NMR experiment.
 86. Themethod according to claim 82 further comprising: subjecting the proteinsample to a RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment (i) to measureand connect the chemical shift values of the α- and β-protons of aminoacid residue i−1, ¹H^(α/β) _(l−1), α- and β-carbons of amino acidresidue i−1, ¹³C^(α/β) _(l−1), ¹⁵N_(l), and ¹H^(N) _(i), and (ii) todistinguish NMR signals for the chemical shift values of ¹H^(β) _(l−1),¹³C^(β) _(l−1), ¹H^(α) _(i−1), and ¹³C^(α) _(l−1) measured by said RD 3DH ^(α/β) C ^(α/β)(CO)NHN NMR experiment from NMR signals for thechemical shift values of ¹H^(ali) _(i−1) and ¹³C^(ali) _(i−1) other than¹H^(α/β) _(i−1) and ¹³C^(α/β) _(i−1) measured by said RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment.
 87. The method according to claim82 further comprising: subjecting the protein sample to a RD 3D H^(α/β),C ^(α/β),N,HN NMR experiment to measure and connect the chemicalshift values of ¹H^(β) _(i), ¹³C^(β) _(i), ¹H^(a) _(i), ¹³C^(α) _(i),¹⁵N_(i), and ¹H^(N) _(l); and obtaining sequential assignments bymatching the chemical shift values of ¹H^(β) _(l), ¹³C^(β) _(l), ¹H^(α)_(i), and ¹³C^(α) _(i) measured by said RD 3D H ^(α/β),C ^(α/β),N,HN NMRexperiment with the chemical shift values of ¹H^(β) _(i−1), ¹³C^(β)_(l−1), ¹H^(α) _(i−1), and ¹³C^(α) _(l−1) measured by said RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment.
 88. The method according to claim82 further comprising: subjecting the protein sample to a 3D HNNCACB NMRexperiment to measure and connect the chemical shift values of ¹³C^(β)_(l), ¹³C^(α) _(i), ¹⁵N_(i), and ¹H^(N) _(l); and obtaining sequentialassignments by matching the chemical shift values of ¹³C^(β) _(l) and¹³C^(α) _(i) measured by said 3D HNNCACB NMR experiment with thechemical shift values of ¹³C^(β) _(l−1) and ¹³C^(α) _(l−1) measured bysaid RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment.
 89. The methodaccording to claim 82 further comprising: subjecting the protein sampleto a RD two-dimensional (2D) HB,CB,(CG,CD),HD NMR experiment to measureand connect the chemical shift values of ¹H^(β) _(i), ¹³C^(β) _(l), anda δ-proton of amino acid residue i with an aromatic side chain, ¹H^(δ)_(i); and obtaining sequential assignments by matching the chemicalshift values of ¹H^(β) _(i) and ¹³C^(β) _(i) measured by said RD 2D HB,CB,(CG, CD), HD NMR experiment with the chemical shift values of ¹H^(β)and ¹³C^(β) measured by said RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment,using said chemical shift values to identify amino acid residue i ashaving an aromatic side chain, and mapping sets of sequentiallyconnected chemical shift values to the amino acid sequence of thepolypeptide chain and locating amino acid residues with aromatic sidechains along said polypeptide chain.
 90. The method according to claim82 further comprising: subjecting the protein sample to a RD 3DH,C,C,H-COSY NMR experiment or a RD 3D H,C,C,H-TOCSY NMR experiment tomeasure and connect the chemical shift values of aliphatic protons ofamino acid residue i, ¹H^(ali) _(i) and aliphatic carbons of amino acidresidue i, ¹³C^(ali) _(i); and obtaining sequential assignments of thechemical shift values of ¹H^(ali) _(l) and ¹³C^(ali) _(i) by (i)matching the chemical shift values of ¹H^(ali) and ¹³C^(ali) measuredusing said RD 3D H,C,C,H-COSY NMR experiment or RD 3D H,C,C,H-TOCSY NMRexperiment with the chemical shift values of ¹H^(β) _(i), ¹³C^(β) _(i),¹H^(α) _(i), and ¹³C^(α) _(i) measured by said RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment and RD 3D HNNCAHA NMR experiment,and (ii) using the chemical shift values of ¹H^(ali) _(i) and ¹³C^(ali)_(i), to identify the type of amino acid residue i.
 91. A method forobtaining assignments of chemical shift values of ¹H, ¹³C and ¹⁵N of aprotein molecule comprising: providing a protein sample; and conductingfour reduced dimensionality (RD) nuclear magnetic resonance (NMR)experiments on the protein sample, wherein (1) a first experiment isselected from the group consisting of a RD three-dimensional (3D) H^(α/β) C ^(α/β)(CO)NHN NMR experiment, a RD 3D HA,CA,(CO),N,HN NMRexperiment, and a RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment forobtaining sequential correlations of chemical shift values; (2) a secondexperiment is selected from the group consisting of a RD 3D HNNCAHA NMRexperiment, a RD 3D H ^(α/β),C ^(α/β),N,HN NMR experiment, and a RD 3DHNN<CO,CA> NMR experiment for obtaining intraresidue correlations ofchemical shift values; (3) a third experiment is a RD 3D H,C,C,H-COSYNMR experiment for obtaining assignments of sidechain chemical shiftvalues; and (4) a fourth experiment is a RD two-dimensional (2D)HB,CB,(CG,CD),HD NMR experiment for obtaining assignments of aromaticsidechain chemical shift values.
 92. The method according to claim 91further comprising: subjecting the protein sample to a RD 2D H,C,H-COSYNMR experiment for obtaining assignments of sidechain chemical shiftvalues.
 93. The method according to claim 91, wherein the firstexperiment is the RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment and thesecond experiment is the RD 3D HNNCAHA NMR experiment.
 94. The methodaccording to claim 93 further comprising: subjecting the protein sampleto a RD 3D HA,CA,(CO),N,HN NMR experiment to distinguish between NMRsignals for ¹H^(α)/¹³C^(α) and ¹H^(β)/¹³C^(β) from said RD 3D H ^(α/β) C^(α/β)(CO)NHN NMR experiment.
 95. The method according to claim 93further comprising: subjecting the protein sample to a RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment to obtain assignments of chemicalshift values of ¹H^(ali) and ¹³C^(ali).
 96. The method according toclaim 93 further comprising: subjecting the protein sample to a RD 3D H^(α/β),C ^(α/β),N,HN NMR experiment to obtain assignments of chemicalshift values of ¹H^(β) and ¹³C^(β).
 97. The method according to claim 93further comprising: subjecting the protein sample to a RD 3D HNN<CO,CA>NMR experiment to obtain assignments of chemical shift values ofpolypeptide backbone carbonyl carbons, ¹³C′.
 98. The method according toclaim 93 further comprising: subjecting the protein sample to a RD 3D H^(α/β) C ^(α/β),CO,HA NMR experiment to obtain assignments of chemicalshift values of polypeptide backbone carbonyl carbons, ¹³C′.
 99. Themethod according to claim 93 further comprising: subjecting the proteinsample to a RD 3D HNN<CO,CA> NMR experiment and a RD 3D H ^(α/β),C^(α/β),CO,HA NMR experiment to obtain assignments of chemical shiftvalues of ¹³C′.
 100. The method according to claim 93 furthercomprising: subjecting the protein sample to a RD 3D H,C,C,H-TOCSY NMRexperiment to obtain assigmnments of chemical shift values of ¹H and ¹³Cof aliphatic sidechains.
 101. The method according to claim 93 furthercomprising: subjecting the protein sample to a RD 3D H,C,C,H-TOCSY NMRexperiment to obtain assignments of chemical shift values of ¹H and ¹³Cof aromatic sidechains.
 102. The method according to claim 93 furthercomprising: subjecting the protein sample to a 3D HNNCACB NMR experimentto obtain assignments of chemical shift values of ¹³C^(β).
 103. Themethod according to claim 93, wherein the first experiment is the RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment and the second experiment is the RD3D HNNCAHA NMR experiment.
 104. The method according to claim 103further comprising: subjecting the protein sample to a RD 3DHA,CA,(CO),N,HN NMR experiment to identify NMR signals for¹H^(α)/¹³C^(α) in said RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment. 105.The method according to claim 103 further comprising: subjecting theprotein sample to a RD 3D H ^(α/β),C ^(α/β),N,HN NMR experiment toobtain assignments of chemical shift values of ¹H^(β) and ¹³C^(β). 106.The method according to claim 103 further comprising: subjecting theprotein sample to a RD 3D HNN<CO,CA> NMR experiment to obtainassignments of chemical shift values of polypeptide backbone carbonylcarbons, ¹³C′.
 107. The method according to claim 103 furthercomprising: subjecting the protein sample to a RD 3D H ^(α/β),C^(α/β),CO,HA NMR experiment to obtain assignments of chemical shiftvalues of polypeptide backbone carbonyl carbons, ¹³C′.
 108. The methodaccording to claim 103 further comprising: subjecting the protein sampleto a RD 3D HNN<CO,CA> NMR experiment and a RD 3D H ^(α/β),C ^(α/β),CO,HANMR experiment to obtain assignments of chemical shift values of ¹³C′.109. The method according to claim 103 further comprising: subjectingthe protein sample to a RD 3D H,C,C,H-TOCSY NMR experiment to obtainassignments of chemical shift values of ¹H and ¹³C of aliphaticsidechains.
 110. The method according to claim 103 further comprising:subjecting the protein sample to a RD 3D H,C,C,H-TOCSY NMR experiment toobtain assignments of chemical shift values of ¹H and ¹³C of aromaticsidechains.
 111. The method according to claim 103 further comprising:subjecting the protein sample to a 3D HNNCACB NMR experiment to obtainassignments of chemical shift values of ¹³C^(β).
 112. The methodaccording to claim 91, wherein the first experiment is the RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment and the second experiment is the RD3D H ^(α/β),C ^(α/β), N,HN NMR experiment.
 113. The method according toclaim 112 further comprising: subjecting the protein sample to a RD 3DHA,CA,(CO),N,HN NMR experiment to identify NMR signals for ¹H^(α) and¹³C^(α) in said RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment.
 114. Themethod according to claim 112 further comprising: subjecting the proteinsample to a RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment to identifyNMR signals for ¹H^(α/β)and ¹³C^(α/β) in said RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment.
 115. The method according to claim112 further comprising: subjecting the protein sample to a RD 3DHNN<CO,CA> NMR experiment to obtain assignments of chemical shift valuesof polypeptide backbone carbonyl carbons, ¹³C′.
 116. The methodaccording to claim 112 further comprising: subjecting the protein sampleto a RD 3D H ^(α/β),C ^(α/β),CO,HA NMR experiment to obtain assignmentsof chemical shift values of polypeptide backbone carbonyl carbons, ¹³C′.117. The method according to claim 112 further comprising: subjectingthe protein sample to a RD 3D HNN<CO,CA> NMR experiment and a RD 3D H^(α/β),C ^(α/β),CO,HA NMR experiment to obtain assignments of chemicalshift values of ¹³C′.
 118. The method according to claim 112 furthercomprising: subjecting the protein sample to a RD 3D H,C,C,H-TOCSY NMRexperiment to obtain assignments of chemical shift values of ¹H and ¹³Cof aliphatic sidechains.
 119. The method according to claim 112 furthercomprising: subjecting the protein sample to a RD 3D H,C,C,H-TOCSY NMRexperiment to obtain assignments of chemical shift values of ¹H and ¹³Cof aromatic sidechains.
 120. The method according to claim 112 furthercomprising: subjecting the protein sample to a 3D HNNCACB NMR experimentto obtain assignments of chemical shift values of ¹³C^(β).
 121. Themethod according to claim 91, wherein the first experiment is the RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment and the second experiment is the RD3D HNN<CO,CA> NMR experiment.
 122. The method according to claim 121further comprising: subjecting the protein sample to a RD 3DHA,CA,(CO),N,HN NMR experiment to identify NMR signals for ¹H^(α) and¹³C^(α) in said RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment.
 123. Themethod according to claim 121 further comprising: subjecting the proteinsample to a RD 3D H ^(α/β) C ^(α/β)(CO)NHN NMR experiment to identifyNMR signals for ¹H^(α/β) and ¹³C^(α/β) in said RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment.
 124. The method according to claim121 further comprising: subjecting the protein sample to a RD 3D H^(α/β),C ^(α/β)CO,HA NMR experiment to obtain assignments of chemicalshift values of polypeptide backbone carbonyl carbons, ¹³C′.
 125. Themethod according to claim 121 further comprising: subjecting the proteinsample to a RD 3D H,C,C,H-TOCSY NMR experiment to obtain assignments ofchemical shift values of ¹H and ¹³C of aliphatic sidechains.
 126. Themethod according to claim 121 further comprising: subjecting the proteinsample to a RD 3D H,C,C,H-TOCSY NMR experiment to obtain assignments ofchemical shift values of ¹H and ¹³C of aromatic sidechains.
 127. Themethod according to claim 121 further comprising: subjecting the proteinsample to a 3D HNNCACB NMR experiment to obtain assignments of chemicalshift values of ¹³C^(β).
 128. The method according to claim 91 furthercomprising: subjecting the protein sample to nuclear Overhauser effectspectroscopy (NOESY) to deduce the tertiary structure of the proteinmolecule.
 129. The method according to claim 91 further comprising:subjecting the protein sample to NMR experiments that measure scalarcoupling constants to deduce the tertiary structure of the proteinmolecule.
 130. The method according to claim 91 further comprising:subjecting the protein sample to NMR experiments that measure residualdipolar coupling constants to deduce the tertiary structure of theprotein molecule.